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Background and Objective Periodontopathogens experience several challenges in the oral cavity that may influence their transcription profile and resulting phenotype. This study evaluated the effect of environmental changes on phenotype and gene expression in a serotype b Aggregatibacter actinomycetemcomitans isolate. Material and Methods Cultures in early exponential phase and at the start of stationary growth phase in microaerophilic and anaerobic atmospheres were evaluated. Cell hydrophobic properties were measured by adherence to n-hexadecane; in addition, adhesion to, and the ability to invade, KB cells was evaluated. Relative transcription of 12 virulence-associated genes was determined by real-time reverse transcritption quantitative PCR. Results The culture conditions tested in this study were found to influence the phenotypic and genotypic traits of A. actinomycetemcomitans. Cells cultured in microaerophilic conditions were the most hydrophobic, reached the highest adhesion efficiency and showed up-regulation of omp100 (which encodes an adhesion) and pga (related to polysaccharide synthesis). Cells grown anaerobically were more invasive to epithelial cells and showed up-regulation of genes involved in host-cell invasion or apoptosis induction (such as apa H, omp29, cag E and cdt B) and in adhesion to extracellular matrix protein ( ema A). Conclusion Environmental conditions of different oral habitats may influence the expression of factors involved in the binding of A. actinomycetemcomitans to host tissues and the damage resulting thereby, and thus should be considered in in-vitro studies assessing its pathogenic potential. [ABSTRACT FROM AUTHOR]

Gonçalves LFH, Fermiano D, Feres M, Figueiredo LC, Teles FRP, Mayer MPA, Faveri M. Levels of Selenomonas species in generalized aggressive periodontitis. J Periodont Res 2012; 47: 711-718. © 2012 John Wiley & Sons A/S Background and Objective: To compare the levels of Selenomonas sputigena and uncultivated/unrecognized Selenomonas species in subgingival biofilms from periodontally healthy subjects and from subjects with generalized aggressive periodontitis. Material and Methods: Fifteen periodontally healthy subjects and 15 subjects with generalized aggressive periodontitis were recruited and their clinical periodontal parameters were evaluated. Nine subgingival plaque samples were collected from each subject and all were individually analyzed for the levels of 10 bacterial taxa, including cultured and uncultivated/unrecognized microorganisms, using the RNA-oligonucleotide quantification technique. Between-group differences in the levels of the test taxa were determined using the Mann-Whitney U-test. Results: Subjects with generalized aggressive periodontitis showed significantly higher mean counts of Porphyromonas gingivalis, S. sputigena and the Mitsuokella sp. Human Oral Taxon (HOT) 131 (previously described as Selenomonas sp. oral clone CS002), while higher mean counts of Actinomyces gerencseriae and Streptococcus sanguinis were found in periodontally healthy subjects ( p < 0.01). Selenomonas sp. HOT 146 was only detected in the generalized aggressive periodontitis group. In the generalized aggressive periodontitis group, the levels of P. gingivalis and S. sputigena were higher in deep sites (probing depth ≥ 5 mm) than in shallow sites (probing depth ≤ 3 mm) ( p < 0.01). Furthermore, in subjects with generalized aggressive periodontitis, sites with probing depth of ≤ 3 mm harbored higher levels of these two species than sites with the same probing depth in periodontally healthy subjects. There were positive correlations between probing depth and the levels of P. gingivalis ( r = 0.77; p < 0.01), S. sputigena ( r = 0.60; p < 0.01) and Selenomonas dianae (previously described as Selenomonas sp. oral clone EW076) ( r = 0.42, p < 0.05). Conclusion: S. sputigena and Mitsuokella sp. HOT 131 may be associated with the pathogenesis of generalized aggressive periodontitis, and their role in the onset and progression of this infection should be investigated further. [ABSTRACT FROM AUTHOR]

Faveri M, Gonçalves LFH, Feres M, Figueiredo LC, Gouveia LA, Shibli JA, Mayer MPA. Prevalence and microbiological diversity of Archaea in peri-implantitis subjects by 16S ribosomal RNA clonal analysis. J Periodont Res 2011; 46: 338-344. © 2011 John Wiley & Sons A/S This study evaluated the prevalence and the molecular diversity of Archaea in the subgingival biofilm samples of subjects with peri-implantitis. 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. 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. 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. [ABSTRACT FROM AUTHOR]

Pinheiro ET, Kawamoto D, Ota-Tsuzuki C, Almeida LRS, Nunes ACR, Longo PL, Wikstrom M, Mayer MPA. Analysis of genotypic variation in genes associated with virulence in Aggregatibacter actinomycetemcomitans clinical isolates. J Periodont Res 2011; 46: 310-317. © 2011 John Wiley & Sons A/S Although certain serotypes of Aggregatibacter actinomycetemcomitans are associated more with aggressive periodontitis than are other serotypes, the correlation between distinct lineages and virulence traits in this species is poorly understood. This study aimed to evaluate the polymorphism of genes encoding putative virulence factors of clinical isolates, and to correlate these findings with A. actinomycetemcomitans serotypes, genotypes and periodontal status of the hosts. Twenty-six clinical isolates from diverse geographic populations with different periodontal conditions were evaluated. Genotyping was performed using pulse-field gel electrophoresis. Polymorphisms in the genes encoding leukotoxin, Aae, ApaH and determinants for serotype-specific O polysaccharide were investigated. The isolates were classified into serotypes a-f, and exhibited three apaH genotypes, five aae alleles and 25 macrorestriction profiles. Two serotype b isolates (7.7%), obtained from Brazilian patients with aggressive periodontitis, were associated with the highly leukotoxic genotype; these isolates showed identical fingerprint patterns and aae and apaH genotypes. Serotype c, obtained from various periodontal conditions, was the most prevalent among Brazilian isolates, and isolates were distributed in two aae alleles, but formed a genetically distinct group based on apaH analysis. Cluster analysis showed a close relationship between fingerprinting genotypes and serotypes/ apaH genotypes, but not with aae genotypes. Apart from the deletion in the ltx promoter region, no disease-associated markers were identified. Non-JP2-like strains recovered from individuals with periodontal disease exhibited considerable genetic variation regarding aae/ apaH genotypes, serotypes and XhoI DNA fingerprints. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The health of family farmers is at risk due to occupational exposure to pesticides. The aims of the current study were to investigate the level of farmers' perception of risks associated with pesticide use and to assess their health condition based on biochemical and immunological tests. Family farmers living in a vegetable-producing region in Southern Brazil were selected to participate in the study. More than 70% of the family farmers were often exposed to more than one type of pesticides; 41.2% were intensively using several pesticides for more than one decade and 74.4% were not using personal protective equipment (PPE) at the time of pesticide handling due to low perception of the risks posed by these chemicals. Enzymatic analysis performed in participants' blood samples showed changes in catalase (CAT) and glutathione reductase (GR) activity, in lipid peroxidation (TBARS) and carbonylated protein levels, as well as in chemoattractant (IL-8) and anti-inflammatory (IL-10) interleukin expression. Low perception of health-related risks posed by pesticides can be attributed to factors such as low schooling and lack of information, which put farmers' health at risk, as evidenced by blood biochemical and immunological changes.

Background: Helicobacter pylori infection in the stomach is associated with gastric and duodenal ulcers, gastric cancers and gastric lymphoma. The organism is transmitted by ingestion, but the oral-oral route and the fecal-oral route are also suggested. The prevalence of infection with H. pylori in developing countries, including Brazil, is higher than in developed countries., Purpose: This study aimed to evaluate the role of the oral cavity as a reservoir of this species, by evaluating the occurrence of H. pylori in supragingival dental plaque and in saliva of Brazilian dyspeptic patients, whether harboring the organism or not in the stomach., Material and Methods: Forty-nine patients reporting dyspeptic symptoms were subjected to oral clinical examination and collection of saliva and supragingival dental plaque samples prior to the endoscopic examination. The detection of H. pylori in oral samples was performed by PCR using 16S rRNA primers. The bacteria were detected in stomach by means of the rapid urease test., Results: Helicobacter pylori was detected in the stomach of 20 of 49 subjects reporting dyspeptic symptoms. The organism was detected in only one supragingival plaque sample, obtained from a patient positive for the urease test in the stomach and in none of the salivary samples., Conclusion: Supragingival dental plaque and saliva may not be relevant reservoirs of H. pylori.

Fimbria encoded by the gene fimA is considered one of the main factors in the colonization of the oral cavity by Porphyromonas gingivalis. Allelic variation in fimA led to the classification of strains of P. gingivalis into six genotypes. The occurrence of P. gingivalis was determined by polymerase chain reaction using 16S rRNA primers in 302 subgingival samples obtained from 102 Brazilian subjects exhibiting different periodontal conditions. Distribution of fimA genotypes was assessed in 146 P. gingivalis positive samples by polymerase chain reaction using primers pairs homologous to the different fimA genes. P. gingivalis was detected in 51 of 57 (89.4%) patients with periodontal attachment loss, in six of 20 gingivitis patients (30.0%) and in two of 25 (8.0%) subjects with a healthy periodontium. Variant type II was the only type detected in 53 sites (39.3%), distributed among 19 periodontitis patients (37.3%) and in one patient with no periodontal destruction. Type Ib was the second most prevalent genotype in periodontitis patients (19.6%). Genotype V was not detected in the studied population. Type IV was the most commonly type found among gingivitis patients, either alone or in combination with other genotypes. Multiple genotypes were detected in nine sites (6.1%). A fimA genotype was not identified in 26 sites (17.8%) of 146 sites positive for P. gingivalis, suggesting that other alleles of fimA not yet sequenced may be prevalent in this population. These data demonstrated that P. gingivalis type II strains followed by type Ib are more prevalent in periodontitis patients from a multiracial population in Brazil, suggesting an increased pathogenic potential of these types.

A cytolethal distending toxin (CDT) found in Actinobacillus actinomycetemcomitans inhibits the eukaryotic cell cycle, which may contribute to the pathogenic potential of the bacterium. The presence of the cdtABC genes and CDT activity were examined in 40 clinical isolates of A. actinomycetemcomitans from Brazil, Kenya, Japan and Sweden. Thirty-nine of 40 cell lysates caused distension of Chinese hamster ovary cells. At least one of the cdt genes was detected in all strains examined. The three cdt genes were detected, by PCR, in 34 DNA samples. DNA from one strain from Kenya did not yield amplicons of the cdtA and cdtB genes and did not express toxic activity. Restriction analysis was performed on every amplicon obtained. PCR-RFLP patterns revealed that the three cdt genes were conserved. These data provided evidence that the cdt genes are found and expressed in the majority of the A. actinomycetemcomitans isolates. Although a quantitative difference in cytotoxicity was observed, indicating variation in expression of CDT among strains, no clear relationship between CDT activity and periodontal status was found.

The occurrence and in vitro antimicrobial sensitivity of isolates of enteric rods and pseudomonads were examined in 80 periodontitis patients, 17 to 58 years of age, in São Paulo, Brazil. Speciation and in vitro antimicrobial susceptibility testing were performed using the BBL Crystal enteric/nonfermenter system and the Etest for amoxicillin/clavulanic acid, ciprofloxacin and doxycycline. A total of 30 strains were isolated from 25 (31.2%) of the study subjects. Pseudomonas aeruginosa occurred in nine patients, Serratia marcescens in seven, and five other species were recovered in lower prevalence. All study organisms demonstrated high susceptibility to ciprofloxacin but exhibited variable susceptibility patterns to the other antimicrobial agents tested. In conclusion, the high occurrence of enteric rods and pseudomonads in these subjects may be important in the pathogenesis of periodontitis, and ciprofloxacin might be the antibiotic of choice to eradicate these pathogens from periodontal pockets.

Objectives: The infection levels of mutans streptococci were investigated during a one-year follow-up in children aged 12 to 30 months attending school nurseries where a sucrose-rich diet was provided., Methods: Oral levels of mutans streptococci obtained from 101 children at baseline and after a one-year follow-up were compared by age, number of teeth, feeding habits, and presence of visible plaque at baseline. Baseline predictors and changes in mutans streptococci levels during the study were compared to caries incidence after one year., Results: Fluctuations in mutans streptococci levels during the follow-up period were not related to feeding habits or presence of visible plaque. Mutans streptococci levels increased after one year among children aged 12 to 24 months, while a significantly higher proportion of those aged 25-30 months showed a decrease in mutans streptococci levels during the study. Multiple logistic regression analysis suggested that high levels of mutans streptococci (> or = 100 cfu) at baseline were associated with a higher caries increment, while reduction in mutans streptococci was negatively associated with caries incidence., Conclusions: Our data suggest that despite early mutans streptococci infection and high exposure to sucrose, mutans streptococci may achieve relatively stable levels after 2 years of age. Heavy colonization by mutans streptococci in an early age was related to an extremely high caries incidence during childhood, while decreasing levels of mutans streptococci can be associated to the decrease in caries activity.

Early mutans streptococci (MS) infection has been associated with higher caries activity in childhood. Since colonization with MS does not always lead to caries activity, additional factors may be involved in MS cariogenicity. For example, MS may differ in virulence traits such as the potential to synthesize glucan polymers from sucrose. In the present study, we tested the hypothesis that caries activity can be associated with variations in virulence factor expression of MS-infecting strains. At baseline, levels of MS obtained by the tongue-blade sampling method, and the presence of visible plaque on upper incisors, were measured in 101 12- to 30-month-old children. Dental caries lesions were diagnosed at baseline and after one year. Caries incidence data were then used to select ten caries-free and nine caries-active children from whom a total of 20 MS fresh isolates was studied. Water-insoluble glucan (WIG) synthesis, final pH, and sucrose-dependent adherence on glass surfaces were measured in these MS isolates. Concentrated culture supernatants were separated in duplicate SDS-PAGE gels, which were then either stained for protein or incubated with 5% sucrose. The intensities of the WIG bands developed in the 5% sucrose PAGE gels and the corresponding protein-stained GTF bands were measured by scanning densitometry. High MS levels (> or = 100 CFU) were associated with high caries incidence (p < 0.01). The presence of visible plaque did not correlate with caries incidence. The intensities of WIG bands were positively correlated with caries incidence (p < 0.05) and with the ability of MS to adhere to glass surfaces (p < 0.05). Analysis of our data suggests that the ability to synthesize WIG is an important virulence factor in initial caries development by increasing MS adherence and accumulation in the plaque of young children.

The aim of the present study was to determine the levels of salivary Streptococcus mutans in Brazilian schoolchildren, and its possible correlation with caries prevalence. A positive correlation between different levels of S. mutans and caries prevalence was observed, individuals with high levels of S. mutans (greater than 100 colonies) had a DMFS significantly higher than those with low or no detectable S. mutans (0, 1-20, 21-40 colonies).