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BACKGROUND: Members of fastidious Granulicatella and Aggregatibacter genera belong to normal oral flora bacteria that can cause serious infections, such as infective endocarditis. Aggregatibacter actinomycetemcomitans has long been implicated in aggressive periodontitis, whereas DNA-based methods only recently showed an association between Granulicatella spp. and dental diseases. As bacterial coaggregation is a key phenomenon in the development of oral and nonoral multispecies bacterial communities it would be of interest knowing coaggregation pattern of Granulicatella species with A. actinomycetemcomitans in comparison with the multipotent coaggregator Fusobacterium nucleatum. The aim was to investigate coaggregation and biofilm formation of Granulicatella elegans and Granulicatella adiacens with A. actinomycetemcomitans and F. nucleatum strains. RESULTS: F. nucleatum exhibited significantly (p < 0.05) higher autoaggregation than all other test species, followed by A. actinomycetemcomitans SA269 and G. elegans. A. actinomycetemcomitans CU1060 and G. adiacens did not autoaggregate. G. elegans with F. nucleatum exhibited significantly (p < 0.05) higher coaggregation than most others, but failed to grow as biofilm together or separately. With F. nucleatum as partner, A. actinomycetemcomitans strains SA269, a rough-colony wild-type strain, and CU1060, a spontaneous smooth-colony laboratory variant, and G. adiacens were the next in coaggregation efficiency. These dual species combinations also were able to grow as biofilms. While both G. elegans and G. adiacens coaggregated with A. actinomycetemcomitans strain SA269, but not with CU1060, they grew as biofilms with both A. actinomycetemcomitans strains. CONCLUSIONS: G. elegans failed to form biofilm with F. nucleatum despite the strongest coaggregation with it. The ability of Granulicatella spp. to coaggregate and/or form biofilms with F. nucleatum and A. actinomycetemcomitans strains suggests that Granulicatella spp. have the potential to integrate into dental plaque biofilms.

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Bacterial biofilms resist host defenses and antibiotics partly because of their decreased metabolism. Some bacteria use proinflammatory cytokines, such as interleukin (IL)-1ß, as cues to promote biofilm formation and to alter virulence. Although one potential bacterial IL-1ß receptor has been identified, current knowledge of the bacterial IL-1ß sensing mechanism is limited. In chronic biofilm infection, periodontitis, Aggregatibacter actinomycetemcomitans requires tight adherence (tad)-locus to form biofilms, and tissue destroying active lesions contain more IL-1ß than inactive ones. The effect of IL-1ß on the metabolic activity of A. actinomycetemcomitans biofilm was tested using alamarBlue™. The binding of IL-1ß to A. actinomycetemcomitans cells was investigated using transmission electron microscopy and flow cytometry. To identify the proteins which interacted with IL-1ß, different protein fractions from A. actinomycetemcomitans were run in native-PAGE and blotted using biotinylated IL-1ß and avidin-HRP, and identified using mass spectroscopy. We show that although IL-1ß slightly increases the biofilm formation of A. actinomycetemcomitans, it reduces the metabolic activity of the biofilm. A similar reduction was observed with all tad-locus mutants except the secretin mutant, although all tested mutant strains as well as wild type strains bound IL-1ß. Our results suggest that IL-1ß might be transported into the A. actinomycetemcomitans cells, and the trimeric form of intracellular ATP synthase subunit ß interacted with IL-1ß, possibly explaining the decreased metabolic activity. Because ATP synthase is highly conserved, it might universally enhance biofilm resistance to host defense by binding IL-1ß during inflammation.

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BACKGROUND: Actinobacillus actinomycetemcomitans is a major pathogen in aggressive periodontitis. Our objectives were to determine the periodontal status and occurrence of A. actinomycetemcomitans in family members of subjects with A. actinomycetemcomitans-positive aggressive periodontitis (AgP) and to evaluate the probability of its intrafamilial transmission. METHODS: Of the 300 subjects screened, 66 (22%) had AgP and A. actinomycetemcomitans. Eleven (probands) of these 66 subjects with AgP met the strict inclusion criteria for the study. The study population consisted of 55 subjects, including probands and their family members (N = 44). Two family groups were formed according to whether the proband was a child (N = 7) or a parent (N = 4). Subgingival samples from all subjects were cultured for A. actinomycetemcomitans, and its clonal types were determined by combining serotype and genotype data for each isolate. RESULTS: Among 42 dentate family members, 16 (38%) exhibited periodontitis and eight (50%) had AgP. Periodontitis was found in nine of 12 (75%) of the dentate parents and six of 17 (35%) siblings of the child probands. A. actinomycetemcomitans was detected in 16 of 31 (52%) family members, i.e., one parent and at least one sibling in six families. The child probands shared A. actinomycetemcomitans clonal types with their parents in five of six (83%) families and with their siblings in three of six (50%) families. In the four parent-proband families, A. actinomycetemcomitans occurred in two spouses and all nine children. The parent probands shared A. actinomycetemcomitans clonal types with their spouses in both families and with their children in three of four families. In all families, the likelihood of intrafamilial transmission of A. actinomycetemcomitans was statistically significant. Members of most families (eight of 11, 73%) also harbored additional clonal types of A. actinomycetemcomitans. CONCLUSION: Parents and siblings of an individual with A. actinomycetemcomitans-positive AgP may have an increased susceptibility to periodontitis and shared and/or other clonal types of oral A. actinomycetemcomitans.

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BACKGROUND: Aggregatibacter actinomycetemcomitans is an oral bacterium associated with aggressively progressing periodontitis. Extracellular release of bacterial outer membrane proteins has been suggested to mainly occur via outer membrane vesicles. This study investigated the presence and conservation of peptidoglycan-associated lipoprotein (AaPAL) among A. actinomycetemcomitans strains, the immunostimulatory effect of AaPAL, and whether live cells release this structural outer membrane lipoprotein in free-soluble form independent of vesicles. RESULTS: The pal locus and its gene product were confirmed in clinical A. actinomycetemcomitans strains by PCR-restriction fragment length polymorphism and immunoblotting. Culturing under different growth conditions revealed no apparent requirement for the AaPAL expression. Inactivation of pal in a wild-type strain (D7S) and in its spontaneous laboratory variant (D7SS) resulted in pleiotropic cellular effects. In a cell culture insert model (filter pore size 0.02 mum), AaPAL was detected from filtrates when strains D7S and D7SS were incubated in serum or broth in the inserts. Electron microscopy showed that A. actinomycetemcomitans vesicles (0.05-0.2 mum) were larger than the filter pores and that there were no vesicles in the filtrates. The filtrates were immunoblot negative for a cytoplasmic marker, cyclic AMP (cAMP) receptor protein. An ex vivo model indicated cytokine production from human whole blood stimulated by AaPAL. CONCLUSION: Free-soluble AaPAL can be extracellularly released in a process independent of vesicles.

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