Microbial composition of atherosclerotic plaques.

OBJECTIVE: The association of infections such as periodontitis with atherosclerotic diseases is well documented. In spite of the high diversity of the human oral microbiota, and its close contact with the circulatory system, few oral species were detected in atherosclerotic plaques. Thus, we attempted to evaluate the microbial diversity of atherosclerotic plaques from patients with different periodontal conditions, submitted to endarterectomy by a broad-range microbial method. MATERIALS AND METHODS: Patients indicated for aorta endarterectomy due to myocardial infarction were recruited for periodontal clinical examination. The microbial diversity of atherosclerotic plaques (n = 35) was evaluated by sequence analysis of bacterial 16S rRNA libraries. RESULTS: Bacterial DNA was detected in 12 endarterectomy specimens (34.3%). Twenty-three bacterial species/phylotypes were identified. Proteobacteria and Firmicutes comprised 78.3% and 21.7% of the identified taxa, respectively. Fifteen (60.9%) phylotypes were reported as yet uncultivable or as yet uncharacterized species. Two uncultured phylotypes were previously detected in the human mouth. The periodontopathogen Aggregatibacter actinomycetemcomitans was detected in seven samples (20%), followed by Pseudomonas species. There was no association between periodontal parameters and detection of A. actinomycetemcomitans or other phylotypes in atherosclerotic plaques. CONCLUSION: Our results suggest a role of the oral microbiota in the development of inflammation in atherogenesis, particularly of A. actinomycetemcomitans.

Analysis of genotypic variation in genes associated with virulence in Aggregatibacter actinomycetemcomitans clinical isolates.

BACKGROUND AND OBJECTIVE: 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. MATERIAL AND METHODS: 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. RESULTS: 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. CONCLUSION: 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.

Aggregatibacter actinomycetemcomitansarcB influences hydrophobic properties, biofilm formation and adhesion to hydroxyapatite / arcB em Aggregatibacter actinomycetmcomitans influencia propriedades hidrofóbicas, formação de biofilme e aderência a hidroxiapatita

The regulation of gene expression in the oral pathogen Aggregatibacter actinomycetemcomitans is still not fully elucidated. ArcAB is a two-component system which allows facultative anaerobic bacteria to sense various respiratory growth conditions and adapt their gene expression accordingly. This study investigated in A. actinomycetemcomitans the role of arcB on the regulation of biofilm formation, adhesion to saliva coated hydroxyapatite (SHA) and the hydrophobic properties of the cell. These phenotypic traits were determined for an A. actinomycetemcomitansarcB deficient type and a wild type strain. Differences in hydrophobic properties were shown at early and late exponential growth phases under microaerobic incubation and at late exponential phase under anaerobiosis. The arcB mutant formed less biofilm than the wild type strain when grown under anaerobic incubation, but displayed higher biofilm formation activity under microaerobic conditions. The adherence to SHA was significantly lower in the mutant when compared with the wild type strain. These results suggest that the transmembrane sensor kinase arcB, in A. actinomycetemcomitans, senses redox growth conditions and regulates the expression of surface components of the bacterial cell related to biofilm formation and adhesion to saliva coated surfaces.

A regulação da expressão gênica do patógeno oral Aggregatibacter actinomycetemcomitans não está completamente descrita. O sistema de dois componentes ArcAB permite que bactérias anaeróbias facultativas percebam diferenças nas condições respiratórias durante sua multiplicação e adaptem a expressão de genes à estas condições. Este estudo investigou em A. actinomycetemcomitans o papel de arcB na regulação da formação de biofilme, aderência à hidroxiapatita recoberta por saliva (SHA) e nas propriedades hidrofóbicas celulares. Estas características fenotípicas foram determinadas para uma linhagem de A. actinomycetemcomitans deficiente em arcB e para uma linhagem selvagem. Foram observadas diferenças nas propriedades hidrofóbicas entre as linhagens quando estas foram cultivadas em ambiente microaerófilo até início e final de fase exponencial e quando foram cultivadas em ambiente anaeróbio até final de fase exponencial. A linhagem arcB mutante formou menos biofilme do que a linhagem selvagem quando estas foram cultivadas sob incubação anaeróbica, porém, apresentou maior formação de biofilme quando a incubação foi realizada em condições de microaerofilia. A aderência à SHA apresentada pela linhagem mutante foi significantemente menor do que a observada pela linhagem selvagem. Estes estudos sugerem que a quinase sensora arcB, em A. actinomycetemcomitans, percebe as condições redox de multiplicação e regula a expressão de componentes de superfície bacterianos relacionados à formação de biofilme e adesão a superfícies recobertas com saliva.

Aggregatibacter actinomycetemcomitans arcB influences hydrophobic properties, biofilm formation and adhesion to hydroxyapatite.

The regulation of gene expression in the oral pathogen Aggregatibacter actinomycetemcomitans is still not fully elucidated. ArcAB is a two-component system which allows facultative anaerobic bacteria to sense various respiratory growth conditions and adapt their gene expression accordingly.This study investigated in A. actinomycetemcomitans the role of ArcB on the regulation of biofilm formation, adhesion to saliva coated hydroxyapatite (SHA) and the hydrophobic properties of the cell. These phenotypic traits were determined for an A. actinomycetemcomitans arcB deficient type and a wild type strain. Differences in hydrophobic properties were shown at early and late exponential growth phases under microaerobic incubation and at late exponential phase under anaerobiosis.The arcB mutant formed less biofilm than the wild type strain when grown under anaerobic incubation, but displayed higher biofilm formation activity under microaerobic conditions. The adherence to SHA was significantly lower in the mutant when compared with the wild type strain. These results suggest that the transmembrane sensor kinase ArcB, in A. actinomycetemcomitans, senses redox growth conditions and regulates the expression of surface components of the bacterial cell related to biofilm formation and adhesion to saliva coated surfaces.

16S rRNA region based PCR protocol for identification and subtyping of Parvimonas micra / PCR baseada na região 16S rRNA para identificação e genotipagem de Parvimonas micra

The present study established a PCR protocol in order to identify Parvimonas micra and to evaluate the intra-species diversity by PCR-RFLP of 16S rRNA partial sequence. The data indicated that the protocol was able to identify this species which could be clustered in five genotypes.

O presente estudo estabeleceu um protocolo de PCR com a finalidade de identificar a espécie Parvimonas micra e avaliar a diversidade intra-espécie utilizando a técnica PCR-RFLP do gene que codifica o rRNA 16S. Os dados indicaram que o protocolo possibilitou a identificação da espécie e a distinção de 5 grupos genotípicos.

16S rRNA region based PCR protocol for identification and subtyping of Parvimonas micra.

The present study established a PCR protocol in order to identify Parvimonas micra and to evaluate the intra-species diversity by PCR-RFLP of 16S rRNA partial sequence. The data indicated that the protocol was able to identify this species which could be clustered in five genotypes.

Genotypic and phenotypic analysis of Streptococcus mutans from different oral cavity sites of caries-free and caries-active children.

INTRODUCTION: Streptococcus mutans exhibits extensive genotypic diversity, but the role of this variation is poorly understood. This study aimed to determine the number and distribution of genotypes of S. mutans isolated from caries-active and caries-free children and to evaluate some of their phenotypic traits. METHODS: Stimulated saliva, tongue surface and biofilms over sound and carious teeth surfaces were sampled from 10 caries-free and 11 caries-active children aged 5-8 years. A total of 339 isolates of S. mutans were genotyped by arbitrarily primed polymerase chain reaction using OPA2 primer. One isolate from each genotype was tested for its acid susceptibility and its ability to form a biofilm. RESULTS: Fifty-one distinct genotypes were determined, one to three genotypes in each oral sample. A single genotype was detected in seven children, whereas the remaining 14 children exhibited two to seven genotypes. There were no significant differences in the number of genotypes detected in caries-free and caries-active children. No correlation was observed between the number of genotypes and the mutans streptococci salivary levels. Five of the six high biofilm-forming genotypes were obtained from caries-active children, although the differences in biofilm formation between isolates from caries-free and caries-active children were not statistically significant. Genotypes with low susceptibility to acid challenge were statistically more frequent among isolates from caries-active children than among those from caries-free children. CONCLUSION: The present data suggested that there were differences in the distribution of genotypes of S. mutans according to the oral site and that S. mutans populations differ in their acid susceptibility and ability to form biofilms, factors allowing their colonization of sucrose-rich environments.

Distribution of fimA genotypes of Porphyromonas gingivalis in subjects with various periodontal conditions.

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.