PknB is a putative transmembrane eukaryotic-type serine/threonine protein kinase (STPK) in the cariogenic bacterium Streptococcus mutans that affects biofilm formation, genetic competence and acid tolerance. PknB contains extracellular penicillin-binding and serine/threonine kinase associated (PASTA) domains predicted to bind the D-alanyl-D-alanine (D-ala-D-ala) dipeptide of unlinked peptidoglycan. D-ala-D-ala elicits responses dependent and independent of the presence of pknB. Biofilm-derived cells of a pknB-deficient mutant (PKNB) exhibited concentration-dependent growth enhancement with D-ala-D-ala, which was not a nutrient response as addition of L-alanine or D-alanine did not give the same results. A total of 77 genes were differentially expressed in PKNB, including 7 with putative functions in fatty acid biosynthesis. PKNB was more sensitive to cell wall- and membrane-targeting antibiotics compared to wild-type. Based on these results, PknB in S. mutans appears to play an important role in cell wall biosynthesis, response to membrane stress and/or regulation of cell membrane composition.

PknB is a putative transmembrane eukaryotic-type serine/threonine protein kinase (STPK) in the cariogenic bacterium Streptococcus mutans that affects biofilm formation, genetic competence and acid tolerance. PknB contains extracellular penicillin-binding and serine/threonine kinase associated (PASTA) domains predicted to bind the D-alanyl-D-alanine (D-ala-D-ala) dipeptide of unlinked peptidoglycan. D-ala-D-ala elicits responses dependent and independent of the presence of pknB. Biofilm-derived cells of a pknB-deficient mutant (PKNB) exhibited concentration-dependent growth enhancement with D-ala-D-ala, which was not a nutrient response as addition of L-alanine or D-alanine did not give the same results. A total of 77 genes were differentially expressed in PKNB, including 7 with putative functions in fatty acid biosynthesis. PKNB was more sensitive to cell wall- and membrane-targeting antibiotics compared to wild-type. Based on these results, PknB in S. mutans appears to play an important role in cell wall biosynthesis, response to membrane stress and/or regulation of cell membrane composition.

No description available.

O objetivo do presente estudo foi testar a hipótese de que polimorfismo na região que codifica o sistema de dois componentes FimSR e seus níveis de transcrição influenciaria expressão de fímbrias e a capacidade de adesão de Porphyromonas gingivalis. Foram avaliadas 21 cepas clínicas e as cepas de referência 33277 (fimbriada) e W83 (não fimbriada). A presença de cápsula foi determinada em 13/21 cepas e de fímbrias em 15/21. Todas as cepas foram capazes de aderir às células (eficiência de 1,2 a 6,1%). Não houve correlação entre os genótipos fimA, presença de fímbrias / cápsula e perfis de macrorestrição por PFGE. Diferenças na transcrição de fimA não podem ser atribuídas a diferenças na região promotora de fimSR. fimA foi regulado positivamente após interação com células epiteliais na maioria das cepas. Os dados indicam que a regulação de fimA é cepa específica. Cepas não-fimbriadas podem apresentar outras estratégias para aderir às células, sugerindo que, além das fímbrias, outras estruturas poderiam desempenhar um papel na interação dessa espécie com as células. / The aim of this study was to test the hypothesis that polymorphism in genes encoding the two-component system FimSR and their transcription levels would influence the expression of fimbriae and adhesion of Porphyromonas gingivalis. We evaluated 21 clinical strains and reference strains 33277 (fimbriate) and W83 (non fimbriated). The presence of a capsule was determined in 13/21 strains and the presence of fimbriae in 15/21. All strains were able to adhere to the cells (efficiency from 1.2 to 6.1%). There was no correlation between genotypes fimA, presence of fimbriae / capsule and macrorestriction profiles by PFGE. Differences in transcription of fimA could not be attributed to differences in the promoter region of fimS. fimA was upregulated after interaction with epithelial cells in most strains. The data indicate that regulation of fimA is strain specific. Non-fimbriated strains may have other strategies to adhere to epithelial cells, suggesting that in addition to fimbriae, other structures could play a role in the interaction of that specie with cells.

O objetivo deste estudo foi determinar a diversidade bacteriana no biofilme subgengival de indivíduos portadores de doença periodontal agressiva (PA). 12 indivíduos com PA e 30 indivíduos com saúde periodontal (PH) foram selecionados. Amostras de placa subgengival foram coletadas de 9 sítios por indivíduo para análise por Hibridação DNA-DNA e de um sítio para a análise clonal 16S. Os patógenos periodontais T. forsythia, P. gingivalis e T. denticola foram encontrados em alta proporção no grupo PA (p<0,001) em comparação ao grupo PH. 120 espécies foram identificadas pela análise clonal de 16SrRNA, sendo 70 destas mais prevalentes. 57% destas espécies são não cultiváveis. Espécies de Selenomonas e Streptococcus foram detectadas em alta prevalencia. Selenomonas sputigena, foi a espécie mais comumente detectada. A microbiota subgengival do grupo PA diferiu marcantemente da do grupo PH. Outras espécies, particularmente do gênero Selenomonas, podem fazer parte da microbiota subgengival em alta proporção em pacientes com PA / The purpose of this study was to determine the bacterial diversity in the subgingival plaque of subjects with aggressive periodontitis (AgP). 12 subject with AgP and 30 periodontally healthy (PH) subjects were selected. Subgingival plaque samples were collected from 9 sites per subject for using in the Checkerboard DNA-DNA technique and one sample by 16S cloning analysis. Periodontal pathogens, such as T. forsythia, P. gingivalis and T. denticola were found in higher proportions AgP groups (p<0.001) than in PH subjects. 120 species were identified by 16S rRNA cloning analyses, therefore 70 species was most prevalent. 57% of the species were not cultivable. Several species of Selenomonas and Streptococcus were found in high prevalence. Selenomonas sputigena, the specie most commonly detected. The subgingival microbiota of AgP markedly differed from PH subjects. Other species, notably species of Selenomonas, may be present in higher proportion in subjects with aggressive periodontitis.

Aggregatibacter (Actinobacillus) actinomycetemcomitans is a Gram-negative coccobacillus of the Pasteurellaceae family. It is implicated in periodontitis, a common low-grade bacterial infection, but it can also cause non-oral infections. The main aim of this project was to identify and characterize in A. actinomycetemcomitans novel cell surface components bearing virulence potential that could contribute to systemic immunoinflammatory burden. We first established and evaluated a method for preparing homogeneous cell suspensions of autoaggregating clinical isolates of A. actinomycetemcomitans. The chosen method is based on a gradual dispersion of bacterial colonies into solution, which generated homogeneous suspensions without losing cell viability or fimbriation. When sera from two patients with A. actinomycetemcomitans-associated infections were used to probe A. actinomycetemcomitans outer membrane protein (OMP) preparations in western blot, strong reactions were found at 17 kDa. Interestingly, antiserum against CsgA, a major subunit of Eschirichia coli curli, also reacted with A. actinomycetemcomitans OMP preparations at 17 kDa size, that is the size of E. coli CsgA, suggesting antigenic crossreactivity. The 17 kDa A. actinomycetemcomitans OMP was subsequently identified as peptidoglycan-associated lipoprotein (PAL; AaPAL) by using immunoproteomics methods. Studies on the pal gene and its gene product showed that they were conserved among the clinical A. actinomycetemcomitans isolates representing all currently known serotypes. AaPAL expression was shown under different nutritional and atmospheric conditions that resembled those in periodontal pockets. PAL deficiency in turn led to pleiotropic effects on the phenotype of A. actinomycetemcomitans, such as cell elongation and decreased growth rate. To purify AaPAL we employed affinity chromatography using anti-AaPAL peptide antibodies. The extensive characterization of the purified AaPAL by SDS-PAGE gel staining and mass spectrometry demonstrated that the final purification product did not contain other bacterial proteins than AaPAL. The protein had not lost its antigenicity during purification, since it was recognized by sera from patients with A. actinomycetemcomitans-associated oral and nonoral infections. AaPAL also appeared to be a strongly immunoreactive antigen in patients with periodontitis whose serum IgG antibodies recognized in western blot a 17 kDa OMP in the parental strain but not in the pal-deficient mutant. In addition to its immunogenicity, AaPAL also induced proinflammatory cytokine and chemokine response from human whole blood as determined by a cytokine antibody array. A cell culture insert model was designed to study how bacterial components could be introduced to the host in infections. The experiments demonstrated that live bacteria released extracellularly free-soluble AaPAL, but also other components, via an unknown outer membrane vesicle-independent mechanism. The immunogenicity and proinflammatory potential of the previously uncharacterized outer membrane lipoprotein of A. actinomycetemcomitans, AaPAL, suggests that it contributes to the pathogenicity of this bacterium. That live A. actinomycetemcomitans cells released free-soluble cell components may represent a new pathogenic mechanism.

O objetivo do presente estudo foi testar a hipótese de que polimorfismo na região que codifica o sistema de dois componentes FimSR e seus níveis de transcrição influenciaria expressão de fímbrias e a capacidade de adesão de Porphyromonas gingivalis. Foram avaliadas 21 cepas clínicas e as cepas de referência 33277 (fimbriada) e W83 (não fimbriada). A presença de cápsula foi determinada em 13/21 cepas e de fímbrias em 15/21. Todas as cepas foram capazes de aderir às células (eficiência de 1,2 a 6,1%). Não houve correlação entre os genótipos fimA, presença de fímbrias / cápsula e perfis de macrorestrição por PFGE. Diferenças na transcrição de fimA não podem ser atribuídas a diferenças na região promotora de fimSR. fimA foi regulado positivamente após interação com células epiteliais na maioria das cepas. Os dados indicam que a regulação de fimA é cepa específica. Cepas não-fimbriadas podem apresentar outras estratégias para aderir às células, sugerindo que, além das fímbrias, outras estruturas poderiam desempenhar um papel na interação dessa espécie com as células. / The aim of this study was to test the hypothesis that polymorphism in genes encoding the two-component system FimSR and their transcription levels would influence the expression of fimbriae and adhesion of Porphyromonas gingivalis. We evaluated 21 clinical strains and reference strains 33277 (fimbriate) and W83 (non fimbriated). The presence of a capsule was determined in 13/21 strains and the presence of fimbriae in 15/21. All strains were able to adhere to the cells (efficiency from 1.2 to 6.1%). There was no correlation between genotypes fimA, presence of fimbriae / capsule and macrorestriction profiles by PFGE. Differences in transcription of fimA could not be attributed to differences in the promoter region of fimS. fimA was upregulated after interaction with epithelial cells in most strains. The data indicate that regulation of fimA is strain specific. Non-fimbriated strains may have other strategies to adhere to epithelial cells, suggesting that in addition to fimbriae, other structures could play a role in the interaction of that specie with cells.

O objetivo deste estudo foi determinar a diversidade bacteriana no biofilme subgengival de indivíduos portadores de doença periodontal agressiva (PA). 12 indivíduos com PA e 30 indivíduos com saúde periodontal (PH) foram selecionados. Amostras de placa subgengival foram coletadas de 9 sítios por indivíduo para análise por Hibridação DNA-DNA e de um sítio para a análise clonal 16S. Os patógenos periodontais T. forsythia, P. gingivalis e T. denticola foram encontrados em alta proporção no grupo PA (p<0,001) em comparação ao grupo PH. 120 espécies foram identificadas pela análise clonal de 16SrRNA, sendo 70 destas mais prevalentes. 57% destas espécies são não cultiváveis. Espécies de Selenomonas e Streptococcus foram detectadas em alta prevalencia. Selenomonas sputigena, foi a espécie mais comumente detectada. A microbiota subgengival do grupo PA diferiu marcantemente da do grupo PH. Outras espécies, particularmente do gênero Selenomonas, podem fazer parte da microbiota subgengival em alta proporção em pacientes com PA / The purpose of this study was to determine the bacterial diversity in the subgingival plaque of subjects with aggressive periodontitis (AgP). 12 subject with AgP and 30 periodontally healthy (PH) subjects were selected. Subgingival plaque samples were collected from 9 sites per subject for using in the Checkerboard DNA-DNA technique and one sample by 16S cloning analysis. Periodontal pathogens, such as T. forsythia, P. gingivalis and T. denticola were found in higher proportions AgP groups (p<0.001) than in PH subjects. 120 species were identified by 16S rRNA cloning analyses, therefore 70 species was most prevalent. 57% of the species were not cultivable. Several species of Selenomonas and Streptococcus were found in high prevalence. Selenomonas sputigena, the specie most commonly detected. The subgingival microbiota of AgP markedly differed from PH subjects. Other species, notably species of Selenomonas, may be present in higher proportion in subjects with aggressive periodontitis.

Archaea ainda não foi reconhecido como patógeno de doença humana. O objetivo deste estudo foi determinar a prevalência, diversidade, níveis e proporções de Archaea no biofilme subgengival de indivíduos com periodontite agressiva (PA) e saúde periodontal (SP). Sessenta indivíduos foram selecionados para este estudo (n=30/grupo). A análise de diversidade foi realizada em 10 indivíduos/grupo. Quatro sítios/indivíduo do grupo PA e 2 sítios/indivíduo do grupo SP foram analisados por qPCR. A freqüência de Archaea foi de 60% dos indivíduos/ 15,2% dos sítios em PA e de 63,3% dos indivíduos/ 15,6% dos sítios em SP (p>0,05). Um a três filotipos foi identificado por amostra. O número de cópias e a proporção de Archaea e Bacteria foram menores no grupo SP do que no grupo PA (p<0,05). Archaea são encontrados no biofilme subgengival de indivíduos com PA e SP. Methanobrevibacter oralis é o filotipo mais prevalente, podendo ser considerado residente da cavidade bucal. A alteração ecológica na microbiota de indivíduos com PA inclui o aumento dos níveis e proporções de Archaea. / Membrers of Archaea domain may be detected in the microbiota of mucous surfaces of human and animals, but their association with diesase have not been yet stablished Some studies have suggested that Archaea domain may be indirectly associated with pathogenesis of periodontitis, since they are found restrict to subgingival sites with severe periodontal destruction. The aim of this study was to determine the prevalence, diversity, levels and proportions of microorganisms of Archaea domain in subgingival biofilm of aggressive periodontitis and periodontally healthy subjects. Thirty generalized aggressive periodontitis (GAgP) and 30 periodontally healthy (PH) subjects were selected. Archaea detection was performed by PCR using domain-specific primers in 9 subgingival samples taken from each subject. Archaea diversity was determined by evaluating a single positive sample per subject, randomly selected from 10 GAgP and 10 PH subjects. Archaeal 16S rRNA gene library were constructed to each sample and the identity of phylotypes were determined for the comparison of unrecognized sequences with gene database. The levels and proportions of Archaea in relation to total microbial load were analysed by quantitative PCR (qPCR) in 4 sites per GAgP subject and 2 sites per PH subject. A total of 540 subgingival samples were analysed to Archaea presence. This domain were detected in 18 GAgP (60%) and in 19 PH (63.3%) subjects. Forty-one (15.2%) and 42 (15.6%) samples were positive for this domain in GAgP and PH subjects, respectively. There was not difference in prevalence of this domain between subjects from GAgP and PH groups, as well as there was not difference in prevalence between sites with different probing depthsin GAgP group. Thenumber of 16S rRNA clones available to identification per sample varies from 33 to 47 in GAgP group, and from 15 to 23 in PH group, with a mean of 42.8+3.9 e 20.2 +2.2, respectively. The analysis of 629 sequencies permits an identification of 1 to 3 phylotypes of Archaea domain per sample. Methanobrevibacter oralis was detected in all archaeal positive samples, being the single detected specie of this domain in 5 subjects from GAgP group, and in 3 subjects from PH group. Methanobacterium curvum/congolense was detected in 3/10 GAgP and 6/10 PH samples, whereas Methanosarcina mazeii was detected in 4/10 samples from both groups. Archaea analysis by qPCR was carried out in 103 sites and 28 GAgP subjects and in 60 sites and 30 PH individuals. Archaea has been detected in 27/28 subjects and in 68% of studied sites in GAgP group and in 26/30 subjects and 58,3% of total analysed sites in PH group. Archaeal and bacterial 16S rRNA mean levels were smaller in PH group than in GAgP group (Mann Whitney, p<0.05). There was no statistic significance of difference in the levels of Archaea in regard to probing depth categories in GAgP group (p>0.05). Moreover, the proportion of Archaea in relation to total microbial load (Archaea + Bacteria) was 0.02% and 0.08% in PH and GAgP group (p<0.05), respectively. These data suggest that Archaea is commonly found in the subgingival biofilm of humans, and that M. oralis may be considered a member of the resident microbiota of subgingival sites. The ecological shift in the microbiota of aggressive periodontitis subjects includes the increase of levels and proportions of Archaea domain.

<p>The aim of this study is to examine the knowledge and experience of the bad breath among high school students in Kristianstad. A further objective was to measure the amount of VSC (Volatile Sulfur Compounds) among those who perceive themselves to have a bad breath. A questionnaire with 21 closed questions was distributed to 120 high school students from Kristianstad municipality who were between the ages of 17-20. The results of this study revealed that most of the students had good knowledge of halitosis. The majority of all participants thought it was important to smell fresh in the mouth and also experienced most of their breath as very good or good. Only a few felt that they had bad breath. However, these persons did think their breath affect their everyday life. Furthermore, they have never felt embarrassed or caught in embarrassing situations due to their breath. The results from the clinical examination was carried out on 8 of the 120 students and was designed to measure the amount of sulphurous gases in the oral cavity which revealed that the average values that emerged in the examination below is the limited values for having a poor breath.</p> / <p>Syftet med denna studie var att undersöka kunskapen och upplevelsen av dålig andedräkt hos gymnasieelever i Kristianstad, ytterligare ett syfte var att mäta mängden VSC (reaktiva svavelföreningar)  hos dem som upplever sig ha dålig andedräkt. En enkät med 21 slutna frågor delades ut till 120 gymnasieelever som var mellan 17-20 år och studerade i Kristanstads kommun. Av resultatet i denna studie framkom att de flesta av gymnasieeleverna hade goda kunskaper om halitosis. Nästan alla 120 som besvarade enkäten tyckte att det var viktigt att lukta fräsch i munnen och dessutom upplevde de flesta sin andedräkt som mycket bra eller bra. Endast ett fåtal (14% ) kände att de ibland hade dålig andedräkt. Hos dessa  påverkades inte andedräkten deras vardagliga liv och de hade aldrig blivit generade eller hamnat i pinsamma situationer på grund av sin andedräkt. Resultatet från den kliniska undersökningen som gjordes på 8 av de 120 elever som besvarade enkäten där mängden av de svavelhaltiga gaserna i munhålan mättes visade att i genomsnitt ligger värden som framkom i undersökningen under gränsvärden för att man skall ha dålig andedräkt.</p>