Gingival thickness and gingival width in children: a cross-sectional study utilizing ultrasonography.

PURPOSE: To measure the gingival phenotype-related features, gingival thickness (GT) and gingival width (GW), in healthy children and to investigate their association between them, with age, gender, tooth-type and arch. METHODS: The gingival sites of 1029 teeth were included from 64 children (36 males and 28 females), with primary and mixed dentition, attending the paediatric dental clinic of Aristotle University, Thessaloniki. GT and GW were measured ultrasonically and with a periodontal probe, respectively. Mixed effects linear regression models were used to evaluate the association of gingival thickness and gingival width with the under-investigation parameters. Spearman's correlation coefficient was used to evaluate correlation between GT and GW. RESULTS: Significantly thicker gingiva is found in posterior teeth compared to anterior teeth, in permanent teeth versus primary teeth and in maxillary teeth in comparison to mandibular teeth (p value < 0.001). Regarding GW, significantly wider gingiva is noted in posterior regions (p value = 0.022) and the maxilla (p value < 0.001). Gender-wise and concerning age GT and GW are not significantly affected. A weak and positive correlation between GT and GW is noted (rho 0.30, p < 0.001). CONCLUSIONS: GT and GW present significant associations with arch and tooth-type. Findings from this study fulfil the further understanding of GT and GW of paediatric patients that are investigated sparsely throughout the literature and demonstrate an accurate, painless and simple method to map the gingiva.

Low prevalence of multi-resistant bacteria in undergraduate dental students; an observational case-control multi-centre study in Europe.

Objective: This study assessed the prevalence of MRSA, ESBL and VRE in students from four dental schools in Europe. Methods: The hand, tongue and nostrils of the students who treated patients (study group) and who did not treat patients (control group) were sampled. After incubation in TSB and subculturing in the presence of 4 µg/ml oxacillin, positive cultures were identified for Staphylococcus aureus by Mannitol salt agar and agglutination tests. The presence of MRSA was confirmed by specific PCR on the species and on the SSCmec genes. ESBL and VRE were isolated using specific CHROMagar and confirmed using antibiotic sensitivity tests. Results: Of the 879 students who participated in this study (454 students which treated patients, 425 controls) a total of 50 students (5.7%) tested positive for a multi-drug resistant bacterium (MDRB); 13 (1.5%) students tested positive for MRSA, 26 (3.0%) for ESBL and 12 (1.4%) for VRE. No statistically significant differences were found between the students who treated patients compared to the control group for any of the MDRB and study centres, excluding MRSA carriage in the Italian student population. The use of antibiotics the year before sampling, was positively associated with the presence of an MDRB (OR 2.0; 95% Confidence Interval 1.10-3.68; p = 0.02). Conclusion: The risk for MDRB carriage and sequential transmission of MDRB for dental health care students and their patients were acceptably low.

Streptococcus mutans, Streptococcus sobrinus and Candida albicans in oral samples from caries-free and caries-active children.

AIM: This was to examine the occurrence of S. mutans, S. sobrinus and C. albicans in dental plaque and saliva from caries-free and caries-active Greek children. METHODS: Saliva and dental plaque samples from 46 caries-free and 51 caries-active 3-to-13-year-old children were examined using selective media for the three microbes. Identification of isolated mutans streptococci (S. mutans and S. sobrinus) was performed with biochemical test and specific DNA probes. The salivary levels of mutans streptococci were additionally determined by a chair-side test (Dentocult® SM strips). RESULTS: The isolation frequencies of S. mutans, S. sobrinus and C. albicans were 66, 11 and 18 %, respectively. Caries-active children harboured more frequently and at significantly higher numbers the specific microbes than caries-free children. A similar pattern was observed with the Dentocult® SM strip scores. No correlation was found between the presence of these microbes and the age or gender of the children. CONCLUSIONS: Caries experience was statistically significantly related to the presence of all three microbes under study, both in dental plaque and saliva.

Transient oral microflora in Greeks attending day centres for the elderly and residents in homes for the elderly.

OBJECTIVE: To examine the isolation frequency and the carriage of yeasts, Enterobacteriaceae, Staphylococcus and Enterococcus species in oral samples from elderly Greeks living alone or in institutions. BACKGROUND: Ageing may promote changes in the oral ecosystem, which lead to colonisation of the mouth by microbes found less commonly or only transiently in younger subjects. Previous studies indicate a geographical variation in the isolation frequency of such bacteria in elderly populations. MATERIALS AND METHODS: Medical and dental records were obtained from 66 attenders at elderly people's day centres (EPDC), and 82 residents of elderly people's homes (EPH), 66-95 years old. Mucosa smear samples were cultured on appropriate media for enumeration of the above species. Microbial identification was performed by conventional microbiological tests. The results were analysed using the Multiple Correspondence Analysis (MCA), ANOVA and other traditional statistical tests. RESULTS: No statistically significant association was found between the place of residence and the wearing of dentures. The isolation frequencies of Staphylococcus aureus, Enterococcus and Enterobacteriaceae species were 21.6, 20.3 and 7.4% respectively. MCA, and further statistical analysis, revealed that the place of residence affected the isolation frequency of years (54.9% in EPH vs. 37.9% in EPDC). Moreover, ANOVA showed that living in EPH increased the carriage of yeasts. CONCLUSIONS: Elderly Greeks exhibit a moderate to high oral carriage of transient bacteria compared with other elderly populations. Living in EPH seems to increase both the isolation frequency and carriage of yeasts.

Cytokine responses of human gingival fibroblasts to Actinobacillus actinomycetemcomitans cytolethal distending toxin.

Actinobacillus actinomycetemcomitans is implicated in the pathogenesis of localized aggressive periodontitis, and has the capacity to express a cytolethal distending toxin (Cdt). Gingival fibroblasts (GF) are resident cells of the periodontium, which can express several osteolytic cytokines. The aims of this study were a) to investigate the role of Cdt in A. actinomycetemcomitans-induced expression of osteolytic cytokines and their cognate receptors in GF and b) to determine if the previously demonstrated induction of receptor activator of NFkappaB ligand (RANKL) by A. actinomycetemcomitans is mediated by these pro-inflammatory cytokines or by prostaglandin E(2) (PGE(2)). A. actinomycetemcomitans clearly induced interleukin (IL)-6, IL-1beta, and to a minimal extent, tumor necrosis factor (TNF)-alpha mRNA expression. At the protein level, IL-6 but not IL-1beta or TNF-alpha expression was stimulated. The mRNA expression of the different receptor subtypes recognizing IL-6, IL-1beta and TNF-alpha was not affected. A cdt-knockout strain of A. actinomycetemcomitans had similar effects on cytokine and cytokine receptor mRNA expression, compared to its parental wild-type strain. Purified Cdt stimulated IL-6, but not IL-1beta or TNF-alpha protein biosynthesis. Antibodies neutralizing IL-6, IL-1 or TNF-alpha, and the PGE(2) synthesis inhibitor indomethacin, did not affect A. actinomycetemcomitans-induced RANKL expression. In conclusion, a) A. actinomycetemcomitans induces IL-6 production in GF by a mechanism largely independent of its Cdt and b) A. actinomycetemcomitans-induced RANKL expression in GF occurs independently of IL-1, IL-6, TNF-alpha, or PGE(2).

The cytolethal distending toxin induces receptor activator of NF-kappaB ligand expression in human gingival fibroblasts and periodontal ligament cells.

Actinobacillus actinomycetemcomitans is associated with localized aggressive periodontitis, a disease characterized by rapid loss of the alveolar bone surrounding the teeth. Receptor activator of NF-kappaB Ligand (RANKL) and osteoprotegerin (OPG) are two molecules that regulate osteoclast formation and bone resorption. RANKL induces osteoclast differentiation and activation, whereas OPG blocks this process by acting as a decoy receptor for RANKL. The purpose of this study was to investigate the effect of A. actinomycetemcomitans on the expression of RANKL and OPG in human gingival fibroblasts and periodontal ligament cells. RANKL mRNA expression was induced in both cell types challenged by A. actinomycetemcomitans extract, whereas OPG mRNA expression remained unaffected. Cell surface RANKL protein was also induced by A. actinomycetemcomitans, whereas there was no change in OPG protein secretion. A cytolethal distending toxin (Cdt) gene-knockout strain of A. actinomycetemcomitans did not induce RANKL expression, in contrast to its wild-type strain. Purified Cdt from Haemophilus ducreyi alone, or in combination with extract from the A. actinomycetemcomitans cdt mutant strain, induced RANKL expression. Pretreatment of A. actinomycetemcomitans wild-type extract with Cdt antiserum abolished RANKL expression. In conclusion, A. actinomycetemcomitans induces RANKL expression in periodontal connective tissue cells. Cdt is crucial for this induction and may therefore be involved in the pathological bone resorption during the process of localized aggressive periodontitis.

Abundant secretion of bioactive interleukin-1beta by human macrophages induced by Actinobacillus actinomycetemcomitans leukotoxin.

Actinobacillus actinomycetemcomitans produces a leukotoxin that selectively kills human leukocytes. Recently, we reported that macrophages are highly sensitive to leukotoxin and that their lysis involves activation of caspase 1. In this study, we show that leukotoxin also induces the production and release of proinflammatory cytokines from human macrophages. The macrophages were challenged with leukotoxin or lipopolysaccharide (LPS) from A. actinomycetemcomitans or LPS from Escherichia coli, and the production and secretion of interleukin-1beta (IL-1beta), IL-6, and tumor necrosis factor alpha (TNF-alpha) were determined at the mRNA and protein levels by reverse transcription-PCR and enzyme-linked immunosorbent assay, respectively. Leukotoxin (1 to 30 ng/ml) induced abundant production and secretion of IL-1beta, while the effects on IL-6 and TNF-alpha production were limited. Leukotoxin (1 ng/ml) caused a 10-times-higher release of IL-1beta than did LPS (100 ng/ml). The secreted IL-1beta was mainly the bioactive 17-kDa protein. At higher concentrations (>30 ng/ml), leukotoxin caused secretion of mainly inactive cytokine, the 31-kDa pro-IL-1beta. The presence of specific antibodies to IL-1beta or of a caspase 1 inhibitor blocked the secretion and production of the cytokine. Supernatants of leukotoxin-challenged macrophages stimulated bone resorption when tested in a mouse calvarial model. The activity could be blocked by an IL-1 receptor antagonist or specific antibodies to IL-1beta. We concluded that A. actinomycetemcomitans leukotoxin can trigger abundant production and secretion of bioactive IL-1beta by human macrophages, which is mediated by activation of caspase 1.

Caspase 1 involvement in human monocyte lysis induced by Actinobacillus actinomycetemcomitans leukotoxin.

Actinobacillus actinomycetemcomitans, an oral bacterium implicated in the etiology of periodontal diseases, produces a leukotoxin that selectively lyses primate neutrophils and monocytes, the major populations of defense cells in the periodontium. Though lysis requires expression of the receptor lymphocyte function-associated molecule 1 (LFA-1) on the cell surface, not all LFA-1-expressing leukocyte populations are equally susceptible to the toxin. In this study, the susceptibility of human leukocytes to leukotoxin-induced lysis is compared to their expression of LFA-1 and the activity of caspase 1. Cytolysis was determined by the activity of lactate dehydrogenase released from peripheral human leukocytes after 1-h exposure to leukotoxin. Monocytes were lysed at leukotoxin concentrations of > or = 5 ng/ml, while the corresponding values for neutrophils and lymphocytes were approximately 10 times greater. Similar LFA-1 expression was found in all susceptible cell populations irrespective of their degree of sensitivity to the toxin. Exposure of monocytes to leukotoxin increased their caspase 1 activity about fivefold within 10 to 20 min. Presence of the caspase 1 inhibitor Ac-YVAD-CMK significantly blocked the leukotoxin-induced lysis of monocytes only. At sublytic concentrations, leukotoxin induced no apoptotic activity in monocytes, as revealed by the lack of caspase 3 activation and DNA fragmentation. Monocytes are the most lysis-sensitive leukocytes for A. actinomycetemcomitans leukotoxin. Their lysis by this toxin depends on caspase 1 activation and proceeds through a process that differs from classical apoptosis.

Serum-mediated release of leukotoxin from the cell surface of the periodontal pathogen Actinobacillus actinomycetemcomitans.

The leukotoxin of the periodontopathogen Actinobacillus actinomycetemcomitans is an important virulence factor that lyses human neutrophils and monocytes and thus, it may enable the bacterium to evade the local host defense. The toxin also induces degranulation of neutrophils and cytokine release in monocytes. To trigger these biological activities, leukotoxin has to be released from the bacterium and diffuse into the periodontal tissues. To date, the conditions found to cause toxin release have been artificial and have included high ion concentration and alkaline conditions. To study the release of the toxin under conditions mimicking the natural environment of the periodontium the ability of human serum to enable leukotoxin release from the bacterial surface was examined. Suspensions of leukotoxic A. actinomycetemcomitans strains were incubated with various concentrations of human serum or serum albumin. The suspensions were centrifuged and the leukotoxin in the supernatants or the cell pellets was detected by gel electrophoresis and immunoblotting. Serum was found to cause the rapid release of leukotoxin from the bacteria in a concentration-dependent manner. Pure albumin exhibited a similar effect. The leukotoxin released was active against human neutrophils. Only a minor proportion of it was associated with membranous vesicles produced by the bacteria. The results indicate that serum, a fluid closely related to the exudate in inflamed periodontal pockets, releases leukotoxin from the cell surface of A. actinomycetemcomitans. The process may enable the diffusion of the toxin from the bacterial biofilm into the surrounding tissues, where it can exert its biological effect.

Release and activation of matrix metalloproteinase 8 from human neutrophils triggered by the leukotoxin of Actinobacillus actinomycetemcomitans.

Matrix metalloproteinase 8 (MMP 8) degrades type I collagen and may be involved in the pathogenesis of periodontitis. Latent MMP 8 is stored in neutrophil granules and can be activated when released extracellularly. The periodontitis-associated bacterium Actinobacillus actinomycetemcomitans produces an RTX-toxin, leukotoxin, that degranulates and lyses human neutrophils. This study deals with the ability of leukotoxic A. actinomycetemcomitans to trigger the release and activation of MMP 8. Whole bacteria of three A. actinomycetemcomitans strains or leukotoxin purified from the highly toxic strain HK 1519 were incubated with human neutrophils. The extracellularly released latent and active forms of MMP 8 were detected by an immunoblot technique using specific antibodies against the protease. The activity of MMP 8 was determined by a collagen degradation assay. All strains induced release and activation of MMP 8. The effect was more pronounced under aerobic than anaerobic conditions and correlated with the leukotoxicity of the strains. Pure leukotoxin also induced MMP 8 release and activation in a concentration-dependent manner. Under aerobic conditions, oxidising substances formed by the neutrophils contributed to the rapid activation of the latent enzyme. Upon anaerobic incubation, the activation was slow and mainly caused by other proteases released during neutrophil degranulation. The activation was totally abolished in the presence of serum, probably due to the serum-protease inhibitors. Compared to the calcium ionophore A 23187, a well-known stimulus of neutrophil degranulation, leukotoxin was a more powerful inducer of MMP 8 release, since it triggered the process at a 1000-fold lower concentration. The present findings reveal a specific mechanism that can be induced by A. actinomycetemcomitans leukotoxin and which may contribute to the degradation of periodontal tissues under certain conditions.

Lack of lipoprotein-dependent effects on the cytotoxic interactions of Actinobacillus actinomycetemcomitans leukotoxin with human neutrophils.

A high odds ratio has been reported for hyperlipidemia and periodontal diseases in humans, and the severity of periodontitis seems to correlate with the hyperlipidemic status of the patients. Early studies indicated that the lipoprotein-containing fraction of the serum enhances the leukotoxic activity of the periodontopathogen Actinobacillus actinomycetemcomitans against human polymorphonuclear leukocytes (PMNL). The protease inhibitors of normal serum account for this enhancement, while delipidated serum has no effect on the leukotoxin-dependent PMNL cytolysis. No information exists for the effect of serum lipoproteins or hyperlipidemic serum. The aim of this study was to evaluate the role of serum lipoproteins in the interaction of the leukotoxin of A. actinomycetemcomitans with human PMNL. Purified leukotoxin was mixed with human PMNL prepared from venous blood of healthy subjects and various varying amounts of hyperlipidemic or delipidated serum, or purified serum lipoproteins. The cytolytic activity of leukotoxin was determined by activity of the cytosol enzyme lactate dehydrogenase released from injured PMNL. The degranulating activity of the toxin was measured through the release of the granule components elastase and lactoferrin. Normal human serum without leukotoxin-neutralizing antibodies caused a 4-fold enhancement of the leukotoxic activity when present at concentrations of 5-10% in the reaction mixture. Serum lipoproteins had no effect when added at concentrations that occur normally in serum. At high concentrations, purified low density and very low-density lipoproteins increased the leukotoxicity of the mixture. Nevertheless, hyperlipidemic serum prepared from a normal serum by the addition of autologous lipoproteins had no influence on the leukotoxin-caused cytolysis compared to the normal serum. Pre-incubation of PMNL for 1 h in hyperlipidemic or delipidated serum had no effect on the leukotoxin-induced degranulation of PMNL. The results indicate that the cytotoxic interactions of A. actinomycetemcomitans leukotoxin against human PMNL are not influenced by the presence of serum lipoproteins.

Proteolytic degradation of oral biofilms in vitro and in vivo: potential of proteases originating from Euphausia superba for plaque control.

This paper deals with enzymatic removal of dental plaque, in vitro as well as in vivo, using proteases from the Antarctic krill shrimp (Euphausia superba), referred to as Krillase. Krillase exhibits both endo- and exopeptidase activity but has no microbicidal effect. In model systems with pure cultures of oral microorganisms. Krillase demonstrated inhibition of microbial adhesion to saliva-coated hydroxyapatite. Furthermore, a protocol for the growth of reproducible in vitro plaque films has been developed, and effects of Krillase on the plaque film were investigated by means of scanning electron microscopy (SEM). The results showed that Krillase efficiently released microorganisms from plaque in vitro, the effect being dependent on the enzymatic activity. The surface energy of the substratum had a minor influence on the formation and removal of plaque in vitro. Ellipsometric studies on the formation and enzymatic removal of a salivary pellicle indicated that the enzymatic effect on plaque may partly depend on degradation of the salivary pellicle. Krillase was also able to remove plaque accumulated on dentures in vivo. Our results demonstrate the potential of Krillase for plaque control, and that these enzymes are worthy of further investigations including clinical studies and work to find a suitable vehicle.

Protease inhibitors, the responsible components for the serum-dependent enhancement of Actinobacillus actinomycetemcomitans leukotoxicity.

Serum enhances the leukotoxic activity of Actinobacillus actinomycetemcomitans against human polymorphonuclear leukocytes (PMNL) by a mechanism that still is unknown. Early attempts to identify the serum components responsible for this enhancement gave no conclusive results, but indicated that the lipoprotein-containing fraction of the serum was involved in the interaction. This study aimed to clarify the role of serum lipoproteins in the leukotoxin interaction, and to identify other serum components involved. The main hypothesis examined was that the leukotoxicity enhancement might depend on serum protease inhibitors that block proteolytic cleavage of leukotoxin by enzymes released from the leukocytes. PMNL were isolated from human peripheral blood and incubated with purified leukotoxin in the presence of serum or purified serum components or lipoprotein-deficient serum. Leukotoxin was also incubated with purified elastase and cathepsin G or with enzyme mixtures from degranulated PMNL. The leukotoxic activity in these mixtures was determined as the extracellular release of lactate dehydrogenase from PMNL. Cleavage of the toxin was showed by gel electrophoresis and Western blot. Morphological changes in PMNL from the above mixtures were examined by electron microscopy. Enzymes from degranulated PMNL cleaved leukotoxin to non-cytotoxic fragments. Elastase and cathepsin G were mainly responsible for the cleavage. Inhibition of leukotoxin degradation was found in the presence of whole serum or of the serum protease inhibitors alpha2-macroglobulin and alpha1-proteinase inhibitor. Under these conditions enhanced PMNL lysis was also observed. A similar enhancement of PMNL lysis was found when PMNL degranulation was blocked by EDTA. On the other hand, lipoprotein-deficient serum had no influence on the leukotoxic activity. The results indicate that the increased leukotoxicity of A. actinomycetemcomitans observed in the presence of human serum is caused by the serum protease inhibitors that counteract proteolytic degradation of leukotoxin. The degradation is caused by enzymes from degranulated PMNL triggered by leukotoxin.

A new bacterial species associated with failed endodontic treatment: identification and description of Actinomyces radicidentis.

OBJECTIVE: This report describes 2 endodontic patients who had persistent signs and symptoms after conventional root canal treatment. The aim of this study was to determine what microorganisms were present in the root canals of the teeth with failed endodontic therapy. STUDY DESIGN: After removal of the root fillings, the canals were sampled by advanced microbiological techniques and the isolates were characterized by various tests. RESULTS: Bacteria, which grew in pure cultures, were isolated in each case. The bacteria were similar to each other and were classified as Actinomyces on the basis of phylogenic and phenotypic evidence. The bacteria were different from others within the genus, thus warranting designation as a new species, Actinomyces radicidentis. CONCLUSIONS: The 2 cases of endodontic failure were infected with A radicidentis, a new Actinomyces species. This bacterium joins a restricted group of other microorganisms that have been associated with failure of root canal treatment.

Characterization of Actinomyces isolates from infected root canals of teeth: description of Actinomyces radicidentis sp. nov.

Two strains of a previously undescribed Actinomyces-like bacterium were recovered in pure culture from infected root canals of teeth. Analysis by biochemical testing and polyacrylamide gel electrophoresis of whole-cell proteins indicated that the strains closely resembled each other phenotypically but were distinct from previously described Actinomyces and Arcanobacterium species. Comparative 16S rRNA gene-sequencing studies showed the bacterium to be a hitherto unknown subline within a group of Actinomyces species which includes Actinomyces bovis, the type species of the genus. Based on phylogenetic and phenotypic evidence, we propose that the unknown bacterium isolated from human clinical specimens be classified as Actinomyces radicidentis sp. nov. The type strain of Actinomyces radicidentis is CCUG 36733.

Cytocidal and apoptotic effects of the ClyA protein from Escherichia coli on primary and cultured monocytes and macrophages.

Cytolysin A (ClyA) is a newly discovered cytolytic protein of Escherichia coli K-12 that mediates a hemolytic phenotype. We show here that highly purified ClyA and ClyA-expressing E. coli were cytotoxic and apoptogenic to fresh as well as cultured human and murine monocytes/macrophages.

Polymorphonuclear leukocyte degranulation induced by leukotoxin from Actinobacillus actinomycetemcomitans.

The ability of leukotoxin from Actinobacillus actinomycetemcomitans to induce release of lysosomal constituents was studied with human polymorphonuclear leukocytes (PMNL). Leukotoxin purified from A. actinomycetemcomitans or bacterial cells of a leukotoxic strain were mixed with human PMNL and the suspension was incubated under anaerobic conditions. Samples were taken at certain time intervals to examine the cell morphology of PMNL by electron microscopy and the extracellular concentrations of the granule components lactoferrin and elastase by enzyme-linked immunosorbent assay (ELISA). Electron microscopy revealed that within 10 min of exposure to leukotoxin, the number of intracellular granules was markedly reduced and the remaining granules were translocated to the periphery in PMNL. At the same time, the extracellular concentrations of lactoferrin and elastase were elevated, while that of the cytosolic enzyme lactate dehydrogenase, an indicator of cell lysis, remained low. The lysosome molecules CD63 and CD66b were also exposed on the PMNL surface, indicating fusion of lysosomes with the plasma membrane. These effects were completely abolished by the addition of anti-leukotoxin serum. Pre-incubation of PMNL with monoclonal antibodies to CD11a and CD18 that recognize alpha- and beta-chains of the LFA-1 integrin, a leukotoxin receptor on PMNL, inhibited the cytolysis, but not the release of granule components. The present results demonstrate the ability of A. actinomycetemcomitans leukotoxin to trigger a rapid release of lysosomal compounds in human PMNL. The release is due to an active process stimulated by the interaction of PMNL with the toxin or toxin-carrying bacteria.

Description of Mogibacterium pumilum gen. nov., sp. nov. and Mogibacterium vescum gen. nov., sp. nov., and reclassification of Eubacterium timidum (Holdeman et al. 1980) as Mogibacterium timidum gen. nov., comb. nov.

A new genus, Mogibacterium, is proposed for anaerobic, non-spore-forming, Gram-positive, rod-shaped bacteria which have been isolated from the periodontal pockets of adult human patients with periodontal disease and infected root canals. The novel isolates, strains D2-18T, BA11a-f and D5-2T, were inert in most of the conventional biochemical tests and phenotypically resemble asaccharolytic Eubacterium species. The protein profiles of whole cells on SDS-PAGE gels and Western immunoblotting reaction analysis distinguished these organisms from type strains belonging to the previously described Eubacterium species. The G + C content of the DNA is 45-46 mol% for Mogibacterium pumilum and 46 mol% for Mogibacterium vescum. The levels of DNA-DNA relatedness of these new species to other Eubacterium species, including Eubacterium limosum, Eubacterium brachy, Eubacterium lentum, Eubacterium nodatum, Eubacterium saphenum, and the more recently proposed Eubacterium minutum and Eubacterium exiguum (reclassified as Slackia exigua), are less than 2%. The DNA-DNA hybridization value between M. pumilum and M. vescum was 30%. Eubacterium timidum exhibited DNA homologies with Mogibacterium species which were low (17 and 18%) but clearly higher than with all the other Eubacterium species. Phylogenetic analysis based on 16S rRNA gene sequences revealed that the closest phylogenetic neighbour of Mogibacterium species was E. timidum, and that these three species represent a novel lineage distinct from the previously described genera of Gram-positive, rod-shaped bacteria. On the basis of phenotypic characteristics and 16S rRNA gene sequence comparisons, it is also proposed that E. timidum is transferred to the genus Mogibacterium gen. nov. as Mogibacterium timidum gen. nov., comb. nov. (type strain ATCC 33093T).

Anaerobic neutrophil-dependent killing of Actinobacillus actinomycetemcomitans in relation to the bacterial leukotoxicity.

The effect of leukotoxin on the interaction of Actinobacillus actinomycetemcomitans with human polymorphonuclear leukocytes (PMNL) was studied under anaerobic conditions with strains able to produce high or low amounts of leukotoxin. PMNL morphology, phagocytosis and killing were examined by transmission electron microscopy and bioassays, respectively. At ratios of > or =25 bacteria/PMNL, a highly toxic A. actinomycetemcomitans strain completely destroyed the PMNL within 7 min, resulting in bacterial survival. Lowering the bacteria/PMNL-ratio enabled phagocytosis and killing of this highly toxic strain. A. actinomycetemcomitans strains with low leukotoxicity were effectively killed by PMNL under any condition. Presence of specific antibodies against A. actinomycetemcomitans or of anti-leukotoxin serum protected PMNL from being injured and allowed phagocytosis to occur. Pre-incubation of the leukotoxic strain with Porphyromonas gingivalis, a bacterium that destroys leukotoxin, abolished lysis of PMNL and inhibited phagocytosis of A. actinomycetemcomitans. The results show that leukotoxin may protect A. actinomycetemcomitans against phagocytosis by human PMNL. The protection occurs at a population level and in relation to the bacterial load. The size of the bacterial population required to counteract phagocytosis is dependent on the leukotoxin production of the strain.

Inhibition of Actinobacillus actinomycetemcomitans leukotoxicity by bacteria from the subgingival flora.

Actinobacillus actinomycetemcomitans produces a pore-forming leukotoxin that lyses human polymorphonuclear leukocytes and monocytes. Certain proteolytic bacteria may coexist with A. actinomycetemcomitans in periodontal pockets. We aimed therefore to examine whether oral bacteria can modify the leukotoxicity of A. actinomycetemcomitans. A total of 55 strains representing 45 bacterial species of the subgingival flora were tested. Each strain was incubated with the highly toxic strain of A. actinomycetemcomitans HK 1519 and the leukotoxic activity of the suspension against human polymorphonuclear leukocytes was determined from the activity of the lactate dehydrogenase released upon lysis of the leukocytes. Porphyromonas gingivalis, Prevotella intermedia, Prevotella nigrescens, Prevotella melaninogenica and Prevotella loeschii inhibited the leukotoxicity of A. actinomycetemcomitans cells as well as the activity of leukotoxin purified from the same strain. The bacterial strains without the ability to block leukotoxic activity also failed to destroy pure leukotoxin even after 5 h of incubation. The proteolytic degradation of leukotoxin by P. gingivalis was mainly dependent on the activity of the enzymes R- and K-gingipains. P. intermedia and P. nigrescens also degraded the leukotoxin by enzymes. The results imply a role of the periodontal microflora in modifying the virulence of A. actinomycetemcomitans by destroying its leukotoxin.