Marcia Pinto Alves Mayer

Microbiological diversity of generalized aggressive periodontitis by 16S rRNA clonal analysis

BACKGROUND/AIM The purpose of this study was to determine the bacterial diversity in the subgingival plaque of subjects with generalized aggressive periodontitis by using culture-independent molecular methods based on 16S ribosomal DNA cloning. METHODS Samples from 10 subjects with generalized aggressive periodontitis were selected. DNA was extracted and the 16S rRNA gene was amplified with the universal primer pairs 9F and 1525R. Amplified genes were cloned, sequenced, and identified by comparison with known 16S rRNA sequences. RESULTS One hundred and ten species were identified from 10 subjects and 1007 clones were sequenced. Of these, 70 species were most prevalent. Fifty-seven percent of the clone (40 taxa) sequences represented phylotypes for which no cultivated isolates have been reported. Several species of Selenomonas and Streptococcus were found at high prevalence and proportion in all subjects. Overall, 50% of the clone libraries were formed by these two genera. Selenomonas sputigena, the species most commonly detected, was found in nine of 10 subjects. Other species of Selenomonas were often present at high levels, including S. noxia, Selenomonas sp. EW084, Selenomonas sp. EW076, Selenomonas FT050, Selenomonas sp. P2PA_80, and Selenomonas sp. strain GAA14. The classical putative periodontal pathogens, such as, Aggregatibacter actinomycetemcomitans, was below the limit of detection and was not detected. CONCLUSION These data suggest that other species, notably species of Selenomonas, may be associated with disease in generalized aggressive periodontitis subjects.

Microbiological profile of untreated subjects with localized aggressive periodontitis

AIM The microbial profile of localized aggressive periodontitis (LAgP) has not yet been determined. Therefore, the aim of this study was to evaluate the subgingival microbial composition of LAgP. MATERIAL AND METHODS One hundred and twenty subjects with LAgP (n=15), generalized aggressive periodontitis (GAgP, n=25), chronic periodontitis (ChP, n=30) or periodontal health (PH, n=50) underwent clinical and microbiological assessment. Nine subgingival plaque samples were collected from each subject and analysed for their content of 38 bacterial species using checkerboard DNA-DNA hybridization. RESULTS Red complex and some orange complex species are the most numerous and prevalent periodontal pathogens in LAgP. The proportions of Aggregatibacter actinomycetemcomitans were elevated in shallow and intermediate pockets of LAgP subjects in comparison with those with GAgP or ChP, but not in deep sites. This species also showed a negative correlation with age and with the proportions of red complex pathogens. The host-compatible Actinomyces species were reduced in LAgP. CONCLUSION A. actinomycetemcomitans seems to be associated with the onset of LAgP, and Porphyromonas gingivalis, Tannerella forsythia, Treponema denticola, Campylobacter gracilis, Eubacterium nodatum and Prevotella intermedia play an important role in disease progression. Successful treatment of LAgP would involve a reduction in these pathogens and an increase in the Actinomyces species.

Association between Caries Prevalence and Clinical, Microbiological and Dietary Variables in 1.0 to 2.5-Year-Old Brazilian Children

The association between caries prevalence and clinical (presence of visible plaque in the labial surfaces of maxillary incisors), microbiological (salivary levels of mutans streptococci) and dietary variables was evaluated in 142 1.0- to 2.5-year-old children attending public day-care nurseries in the city of Piracicaba – São Paulo. A significant difference in caries prevalence was observed between those children with and without visible plaque (χ2 = 12.08, p < 0.001). The mean ds (decayed surfaces) was significantly higher in children with visible plaque on the maxillary incisors than in children without it (p < 0.001). Mutans streptococci were detected in 114 (80.3%) of the children. A significantly higher caries prevalence was observed in children with high levels of mutans streptococci compared to children with low levels (χ2 = 28.67, p < 0.001). The mean ds was significantly higher in children with levels of mutans streptococci greater than 50 CFU when compared to children with 0 CFU or 1–50 CFU of mutans streptococci (p < 0.05). Children who were either never breast-fed or only until 3 months exhibited a significantly higher caries prevalence than those breast-fed for a longer time (χ2 = 4.11, p < 0.05). A significantly higher caries prevalence was also observed between children that used bottle containing milk with sucrose and cereal than children using bottle with milk with or without sucrose (χ2 = 6.24, p < 0.05). Children who started to eat salty meals at or after 7 months of age showed a significant higher caries prevalence than children who started earlier (χ2 = 10.30, p < 0.01). These data support the evidence of an association between caries prevalence in young children and mutans streptococci levels, clinical and dietary factors.

Role of periodontal pathogenic bacteria in RANKL-mediated bone destruction in periodontal disease

Abstract Accumulated lines of evidence suggest that hyperimmune responses to periodontal bacteria result in the destruction of periodontal connective tissue and alveolar bone. The etiological roles of periodontal bacteria in the onset and progression of periodontal disease (PD) are well documented. However, the mechanism underlying the engagement of periodontal bacteria in RANKL-mediated alveolar bone resorption remains unclear. Therefore, this review article addresses three critical subjects. First, we discuss earlier studies of immune intervention, ultimately leading to the identification of bacteria-reactive lymphocytes as the cellular source of osteoclast-induction factor lymphokine (now called RANKL) in the context of periodontal bone resorption. Next, we consider 1 the effects of periodontal bacteria on RANKL production from a variety of adaptive immune effector cells, as well as fibroblasts, in inflamed periodontal tissue and 2 the bifunctional roles (upregulation vs. downregulation) of LPS produced from periodontal bacteria in a RANKL-induced osteoclast-signal pathway. Future studies in these two areas could lead to new therapeutic approaches for the management of PD by down-modulating RANKL production and/or RANKL-mediated osteoclastogenesis in the context of host immune responses against periodontal pathogenic bacteria.

Diversity and quantitative analysis of Archaea in aggressive periodontitis and periodontally healthy subjects

AIM To investigate the diversity, levels and proportions of Archaea in the subgingival biofilm of generalized aggressive periodontitis (GAgP; n=30) and periodontally healthy (PH; n=30) subjects. MATERIALS AND METHODS Diversity was determined by sequencing archaeal 16S rRNA gene libraries from 20 samples (10/group). The levels and proportions of Archaea were analysed by quantitative PCR (qPCR) in four and two samples/subject in GAgP and PH groups, respectively. RESULTS Archaea were detected in 27/28 subjects and 68% of the sites of the GAgP group, and in 26/30 subjects and 58.3% sites of the PH group. Methanobrevibacter oralis was found in all 20 samples studied, Methanobacterium curvum/congolense in three GAgP and six PH samples, and Methanosarcina mazeii in four samples from each group. The levels and proportions of Archaea were higher in GAgP than in PH, whereas no differences were observed between the two probing depth category sites from the GAgP group. CONCLUSION Archaea were frequently found in subjects with periodontal health and GAgP, especially M. oralis. However, the higher levels and proportions (Archaea/total prokaryotes) of this domain observed in GAgP in comparison with PH subjects indicate a possible role of some of these microorganisms as an environmental modifier in GAgP.

Quantification of Porphyromonas gingivalis and fimA genotypes in smoker chronic periodontitis

AIM Porphyromonas gingivalis fimA genotypes were associated with virulence factors in vitro, but little evidence of an association with disease severity were shown in humans. We aimed to correlate levels of P. gingivalis fimA genotypes II and IV and probing depth in smoker-chronic periodontitis subjects. MATERIAL AND METHODS One hundred and sixty eight subgingival samples of 20 smokers non-treated chronic periodontitis subjects obtained from sites with different probing depths [shallow (< or =3 mm), intermediate (4-6 mm), deep (> or =7 mm)] were analysed by real-time PCR for P. gingivalis and genotypes fimA II and IV. RESULTS P. gingivalis and fimA IV were detected in all subjects, whereas fimA II was detected in 18 subjects (90%). One hundred and fifty two sites (90.5%) harboured P. gingivalis. Genotypes II and IV were detected in 28% and 69.6% of sites, respectively. The proportions of genotypes II and IV in relation to P. gingivalis levels were similar in shallow, intermediate and deep probing sites (2.4%, 4.6%, 1.4% for genotype II and 15.5%, 17.7%, 11.7% for genotype IV, respectively), indicating that other non-tested genotypes were more abundant. Increased levels of genotype IV were associated with increasing probing depth, but not of genotype II. CONCLUSIONS The data suggested an association between P. gingivalis genotype fimA IV and disease severity in smoker-chronic periodontitis subjects.

In vitro analysis of inhibitory effects of the antibacterial monomer MDPB-containing restorations on the progression of secondary root caries

OBJECTIVE This study aimed to analyze in vitro inhibitory effects of restorative materials containing the antibacterial monomer 12-methacryloyloxydodecylpyridinium bromide (MDPB) on the formation of artificial secondary root caries lesions. METHODS Class V cavities (2mmx2mm) were prepared in 75 human root fragments. Specimens were randomly divided into five groups (n=15 fragments per group) and restored as follows: (I) MDPB-free adhesive system+MDPB-free composite (negative control); (II) resin modified glass ionomer (RM-GIC; positive control); (III) MDPB-free adhesive system+MDPB-containing composite (2.83% MDPB); (IV) MDPB-containing adhesive system+MDPB-free composite; (V) MDPB-containing adhesive system+MDPB-containing composite. Artificial secondary root caries lesions were produced by a biological artificial caries challenge. The restored specimens were immersed into a culture medium containing Streptococcus mutans and sucrose for 15 days. Histological slices (80+/-20 microm) of the specimens were used for measuring the mean depths of the artificial lesions produced in both margins of the restorations using polarized light microscopy. Results were expressed in percentage related to the mean depth of the negative control, considered 100%. Data were compared by ANOVA followed by the Tukeys test (p< or =0.05). RESULTS The depths of lesions adjacent to cavities filled with RM-GIC (GII; 85.17+/-15.2%) were significantly (p<0.01) shallower than those adjacent to restorations with MDPB-free composite (GI; 100.00+/-10.04%), despite the presence of MDPB in the adhesive system (GIV; 101.95+/-21.32%). The depths of lesions adjacent to cavities restored with MDPB-containing composite (GIII; 82.68+/-12.81% and GV; 85.65+/-15.42%), despite the adhesive system used, were similar to those of RM-GIC (GII). Mean lesions depths in these groups decreased from 13% (GV) to 17% (GIII) in relation to the negative control (GI). CONCLUSIONS MDPB-containing composite inhibits the progression of artificial secondary root caries lesions regardless of adhesive systems.

Prevalence and microbiological diversity of Archaea in peri-implantitis subjects by 16S ribosomal RNA clonal analysis

BACKGROUND AND OBJECTIVE This study evaluated the prevalence and the molecular diversity of Archaea in the subgingival biofilm samples of subjects with peri-implantitis. MATERIAL AND METHODS Fifty subjects were assigned into two groups: Control (n = 25), consisting of subjects with healthy implants; and Test (n = 25), consisting of subjects with peri-implantitis sites, as well as a healthy implant. In the Test group, subgingival biofilm samples were taken from the deepest sites of the diseased implant. In both groups, subgingival biofilm was collected from one site with a healthy implant and from one site with a periodontally healthy tooth. DNA was extracted and the 16S ribosomal RNA gene was amplified with universal primer pairs for Archaea. Amplified genes were cloned and sequenced, and the phylotypes were identified by comparison with known 16S ribosomal RNA sequences. RESULTS In the Control group, Archaea were detected in two and three sites of the implant and the tooth, respectively. In the Test group, Archaea were detected in 12, 4 and 2 sites of diseased implants, healthy implants and teeth, respectively. Diseased implants presented a significantly higher prevalence of Archaea in comparison with healthy implants and natural teeth, irrespective of group. Over 90% of the clone libraries were formed by Methanobrevibacter oralis, which was detected in both groups. Methanobacterium congelense/curvum was detected in four subjects from the Test group and in two subjects from the Control group. CONCLUSION Although M. oralis was the main species of Archaea associated with both healthy and diseased implant sites, the data indicated an increased prevalence of Archaea in peri-implantitis sites, and their role in pathogenesis should be further investigated.

Synergistic Anti-Inflammatory Activity of the Antimicrobial Peptides Human Beta-Defensin-3 (hBD-3) and Cathelicidin (LL-37) in a Three-Dimensional Co-Culture Model of Gingival Epithelial Cells and Fibroblasts

Given the spread of antibiotic resistance in bacterial pathogens, antimicrobial peptides that can also modulate the immune response may be a novel approach for effectively controlling periodontal infections. In the present study, we used a three-dimensional (3D) co-culture model of gingival epithelial cells and fibroblasts stimulated with Aggregatibacter actinomycetemcomitans lipopolysaccharide (LPS) to investigate the anti-inflammatory properties of human beta-defensin-3 (hBD-3) and cathelicidin (LL-37) and to determine whether these antimicrobial peptides can act in synergy. The 3D co-culture model composed of gingival fibroblasts embedded in a collagen matrix overlaid with gingival epithelial cells had a synergistic effect with respect to the secretion of IL-6 and IL-8 in response to LPS stimulation compared to fibroblasts and epithelial cells alone. The 3D co-culture model was stimulated with non-cytotoxic concentrations of hBD-3 (10 and 20 µM) and LL-37 (0.1 and 0.2 µM) individually and in combination in the presence of A. actinomycetemcomitans LPS. A multiplex ELISA assay was used to quantify the secretion of 41 different cytokines. hBD-3 and LL-37 acted in synergy to reduce the secretion of GRO-alpha, G-CSF, IP-10, IL-6, and MCP-1, but only had an additive effect on reducing the secretion of IL-8 in response to A. actinomycetemcomitans LPS stimulation. The present study showed that hBD-3 acted in synergy with LL-37 to reduce the secretion of cytokines by an LPS-stimulated 3D model of gingival mucosa. This combination of antimicrobial peptides thus shows promising potential as an adjunctive therapy for treating inflammatory periodontitis.

Histomorphometric and Microbiological Assessment of Photodynamic Therapy as an Adjuvant Treatment for Periodontitis: A Short-Term Evaluation of Inflammatory Periodontal Conditions and Bacterial Reduction in a Rat Model

OBJECTIVE The aim of this study was to investigate the short-term effects of photodynamic therapy (PDT) in periodontal tissue when it is used as an adjuvant treatment for periodontitis. BACKGROUND DATA PDT has been used as an adjuvant in the combat of local infections, such as periodontitis, and combines a photosensitizer (PS) with a light source to induce reactive oxygen species (ROS) and kill microbial cells. METHODS Fifty healthy male rats were used in this study. Periodontitis was induced by placing a cotton ligature around the upper left second molar in a subgingival position. Posterior maxillas were removed and histologically prepared with hematoxylin & eosin (H&E) staining techniques. PDT was performed with a diode laser (λ=660 nm) with an output power of 100 mW. Methylene blue aqueous solution (100 μM) was used as the PS while control group used phosphate buffered saline (PBS). Collagen organization, inflammatory infiltrate, and bone loss were evaluated. Bacterial samples were collected before and immediately after treatment to determine bacterial reduction. RESULTS The experimental group that was treated with PDT presented better periodontal healing, as measured by collagen organization, inflammatory infiltrate, and bone loss. Significant bacterial reduction was achieved following treatment with or without PDT compared to control, with a higher microbial reduction observed in the PDT group. CONCLUSIONS PDT used as an adjuvant treatment showed effective short-term control of periodontitis infection.