Dental and periodontal health, and microbiological and salivary conditions in patients with or without diabetes undergoing haemodialysis.

OBJECTIVE: The aim of this cross-sectional study was to evaluate the dental and periodontal health, as well as the microbiological and salivary conditions, of patients with and without diabetes mellitus (DM) who are receiving haemodialysis. METHODS: One-hundred and fifty-nine haemodialysis patients were included and divided into groups according to the pre-existing diabetes status: DM or no DM. The oral examination included dental findings and assessment of the periodontal situation. The periodontal condition was classified as healthy/mild, moderate or severe periodontitis. Subgingival biofilm samples were analysed using the polymerase chain reaction. The salivary diagnostics included measurement of unstimulated and stimulated salivary flow, pH and buffer capacity. Statistical analyses used Fisher's test, the t-test and the Mann-Whitney U-test (α = 5%). RESULTS: The dental findings showed no significant difference between patients with and without DM (P = 0.44). The prevalence of periodontitis was high (96% in patients with DM and 97% in patients who did not have DM) and there was no significant difference between the groups (P = 0.71). There was a higher prevalence of Porphyromonas gingivalis, Parvimonas micros, Eubacterium nucleatum and Capnocytophaga spp. in patients without DM (P < 0.05). The salivary pH was significantly higher in patients without DM (P < 0.01). CONCLUSION: While differences in the prevalence of periodontal pathogenic bacteria and in the salivary pH were detected between the groups, the dental and periodontal status was comparable between patients with and without DM. Accordingly, DM appears to have no decisive influence on the oral health in patients treated with haemodialysis who have well-controlled diabetes.

Bloom and bust: intestinal microbiota dynamics in response to hospital exposures and Clostridium difficile colonization or infection.

BACKGROUND: Clostridium difficile infection (CDI) is the leading infectious cause of nosocomial diarrhea. Hospitalized patients are at increased risk of developing CDI because they are exposed to C. difficile spores through contact with the hospital environment and often receive antibiotics and other medications that can disrupt the integrity of the indigenous intestinal microbiota and impair colonization resistance. Using whole metagenome shotgun sequencing, we examined the diversity and composition of the fecal microbiota in a prospective cohort study of 98 hospitalized patients. RESULTS: Four patients had asymptomatic C. difficile colonization, and four patients developed CDI. We observed dramatic shifts in the structure of the gut microbiota during hospitalization. In contrast to CDI cases, asymptomatic patients exhibited elevated relative abundance of potentially protective bacterial taxa in their gut at the onset of C. difficile colonization. Use of laxatives was associated with significant reductions in the relative abundance of Clostridium and Eubacterium; species within these genera have previously been shown to enhance resistance to CDI via the production of secondary bile acids. Cephalosporin and fluoroquinolone exposure decreased the frequency of Clostridiales Family XI Incertae Sedis, a bacterial family that has been previously associated with decreased CDI risk. CONCLUSIONS: This study underscores the detrimental impact of antibiotics as well as other medications, particularly laxatives, on the intestinal microbiota and suggests that co-colonization with key bacterial taxa may prevent C. difficile overgrowth or the transition from asymptomatic C. difficile colonization to CDI.

Distribution of secretory inhibitor of platelet microbicidal protein among anaerobic bacteria isolated from stool of children with diarrhea.

AIM: To study the secretory inhibitor of platelet microbicidal protein (SIPMP) phenotypes of faecal anaerobic isolates from patients with diarrhea. METHODS: Faecal isolates of anaerobic bacteria (B. fragilis, n = 42; B. longum, n = 70; A. israelii, n = 21; E. lentum, n = 12) from children with diarrhea were tested. SIPMP production was tested by inhibition of platelet microbicidal protein (PMP) bioactivity against B. subtilis and was expressed as percentage of inhibition of PMP bactericidal activity. RESULTS: Among anaerobic isolates 80% of B. longum strains, 85.7% of A. israelii strains, 50% of E. lentum strains and 92.86% of B. fragilis strains were SIPMP-positive. The isolated anaerobic organisms demonstrated SIPMP production at a mean level of 13.8% +/- 0.7%, 14.7% +/- 1.8%, 3.9% +/- 0.9% (P < 0.05) and 26.8% +/- 7.5% (P < 0.05) for bifidobacteria, A. israelii, E. lentum and B. fragilis, respectively. CONCLUSION: Data from the present study may have significant implications in understanding the pathogenesis of microecological disorders in the intestine, as well as for future improvement in the prevention and therapy of anaerobe-associated infections.

Association of Eubacterium nodatum and Treponema denticola with human periodontitis lesions.

BACKGROUND: The purpose of the present investigation was to compare the levels, proportions and percentage of sites colonized by 40 bacterial species in subgingival plaque samples from periodontally healthy subjects and patients with chronic periodontitis to seek possible pathogens other than the consensus pathogens Porphyromonas gingivalis and Tannerella forsythia. METHOD: Subgingival plaque samples were taken from the mesial aspect of each tooth in 635 subjects with chronic periodontitis and 189 periodontally healthy subjects. The samples were individually analyzed for their content of 40 bacterial species using checkerboard DNA-DNA hybridization (total samples = 21,832). Mean counts, % DNA probe counts and percentage of sites colonized at >10(5) were determined for each species in each subject and then averaged in each clinical group. Significance of difference between groups was determined using the Mann-Whitney test. Association between combinations of species and periodontal status was examined by stepwise logistic regression analysis. Analyses were repeated using a subset of subjects from both clinical groups who had proportions of P. gingivalis plus T. forsythia less than the median (4.42%) found in periodontally healthy subjects. All analyses were adjusted for multiple comparisons. RESULTS: For the 824 subjects the consensus pathogens P. gingivalis and T. forsythia as well as Eubacterium nodatum and Treponema denticola had significantly higher mean counts, proportions and percentage of sites colonized in samples from subjects with periodontitis than from periodontally healthy subjects. There were significantly more Capnocytophaga gingivalis, Streptococcus gordonii and Veillonella parvula in periodontally healthy subjects. E. nodatum, T. denticola, Streptococcus oralis, Streptococcus intermedius, Fusobacterium nucleatum ssp. vincentii all had higher counts and proportions in diseased than healthy subjects who had low proportions of P. gingivalis and T. forsythia. Logistic regression analysis indicated that the same species groups were associated with disease status after adjusting for the proportions of the other species. CONCLUSIONS: This investigation confirmed the strong association of P. gingivalis and T. forsythia with chronic periodontitis and emphasized a strong association of E. nodatum and T. denticola with periodontitis whether in the presence or absence of high levels of the consensus pathogens. Other species, including S. oralis, Eikenella corrodens, S. intermedius and F. nucleatum ssp. vincentii, were associated with disease when P. gingivalis and T. forsythia were present in low proportions.

Detection frequency of periodontitis-associated bacteria by polymerase chain reaction in subgingival and supragingival plaque of periodontitis and healthy subjects.

The aim of this study was to compare the detection frequencies of 25 bacterial species in subgingival and supragingival plaque of 18 untreated periodontitis subjects and 12 periodontally healthy subjects. Genomic DNA was extracted from subgingival and supragingival plaque samples, and bacterial detection was performed by polymerase chain reaction of the 16S rRNA genes. Fourteen bacteria showed no relationship with periodontitis, and 11 of these 14 species were frequently detected (> or =50%) in subgingival plaque in both periodontitis and healthy subjects. Nine bacteria such as Eubacterium saphenum, Prevotella intermedia, and Treponema denticola seemed to be related to periodontitis; their detection frequencies in subgingival plaque samples were higher in periodontitis than in healthy subjects, but these differences were not statistically significant by multiple comparisons (0.002< or =P<0.05). Two species (Mogibacterium timidum and Porphyromonas gingivalis) were detected significantly more frequently in subgingival plaque of periodontitis subjects than of healthy subjects (P<0.002), with P. gingivalis being detected only in periodontitis subjects, suggesting that these two species are closely related to periodontitis. There were no significant differences in the detection frequencies of the 25 bacteria between subgingival and supragingival plaque, suggesting that the bacterial flora of supragingival plaque reflects that of subgingival plaque.

Pseudoramibacter alactolyticus in primary endodontic infections.

A nested polymerase chain reaction (PCR)-based method was used to directly survey samples taken from primary endodontic infections for the occurrence of Pseudoramibacter alactolyticus. Identification by nested PCR was performed in root-canal samples from teeth associated with asymptomatic periradicular lesions or acute apical periodontitis, and in pus samples from acute periradicular abscesses. DNA was extracted from the samples and initially amplified using universal 16S rDNA primers. A second round of amplification used the first PCR products to detect a specific fragment of P. alactolyticus 16S rDNA. P. alactolyticus was detected in 76% of root-canal samples from teeth showing asymptomatic periradicular lesions, in 60% of samples taken from root canals associated with acute apical periodontitis, and in 32% of pus samples aspirated from acute periradicular abscesses. No significant association of this species with clinical symptoms was observed (p > 0.01). In general, P. alactolyticus occurred in 56% of samples taken from infections of endodontic origin. The high prevalence of P. alactolyticus in infections of endodontic origin as detected by nested PCR in this study, and its apparent pathogenicity, particularly in mixed infections, indicate that this bacterial species is a candidate endodontic pathogen that can participate in the etiology of different forms of periradicular diseases.

Eubacterium callanderi bacteremia: report of the first case.

Eubacterium callanderi is an environmental anaerobic rod-shaped bacterium first isolated in 1998 from an industrial anaerobic digester. We report on the first clinical isolate of E. callanderi, which was recovered from the blood of a patient with a bladder carcinoma. Identification of the organism was made by cell fatty acid chromatographic analysis and 16S rRNA gene sequencing.

Enzyme degradation and proinflammatory activity in arthritogenic and nonarthritogenic Eubacterium aerofaciens cell walls.

Two almost-identical strains of Eubacterium aerofaciens isolated from the normal human gut flora were used. The cell wall (CW) of one strain with a peptidoglycan (PG) type A4alpha induces chronic arthritis in the rat after a single intraperitoneal injection, whereas CW of the other with PG type A4beta induces only a transient acute arthritis. The CW of the arthritogenic E. aerofaciens was a twofold-more-potent stimulator of the proinflammatory cytokines tumor necrosis factor alpha (TNF-alpha) and monocyte chemoattractant protein 1 (MCP-1) than the nonarthritogenic CW. After degradation with mutanolysin, the capacity of the arthritogenic PG to stimulate production of TNF-alpha and MCP-1 was significantly increased, whereas that of the nonarthritogenic PG was significantly decreased. In other words, after enzyme degradation the arthritogenic PG had a four- to fivefold-stronger stimulatory capacity than that of the enzyme-treated nonarthritogenic PG. These findings indicate that the arthritogenicity of CW or a PG is not dependent on the enzyme resistance alone but also on how the PG fragments released by enzyme degradation stimulate the production of proinflammatory cytokines.

Intraperitoneal immune cell responses to Eubacterium saphenum in mice.

Oral asaccharolytic Eubacterium saphenum, which are newly isolated gram-positive rods and one of the predominant microorganisms in human periodontal pockets, were injected intraperitoneally in mice to elucidate their pathogenicity in periodontal diseases. Infiltrating immune cells in the peritoneal exudate were quantitated and intracellular T cell (CD4+/CD8+/gammadelta+) production of cytokines IL-4 and IFN-gamma which are related to cellular and humoral immunity, respectively, was determined. Neutrophils appeared first in peritoneal exudates, followed by macrophages and lymphocytes, after the injection of either E. saphenum or Porphyromonas gingivalis. Intracellular IL-4+ and IFN-gamma+ gammadelta T cells were detected in the exudates after the injection of E. saphenum (4.6 +/- 0.8% and 10.1 +/- 1.4%, respectively) and P. gingivalis (5.3 +/- 1.6% and 10.1 +/- 2.1%, respectively). The intracellular production of IL-4/IFN-gamma in CD4+/CD8+ T cells was rather low indicating that the main response was from gammadelta T cells which initiated the immune reactions in mouse peritoneal cavities after injection of E. saphenum or P. gingivalis. Serum IgG and IgM levels were elevated in animals injected with E. saphenum and similarly with P. gingivalis. The present study showed that with slight differences, similar modes of cell response and cytokine and Ig production were observed after intraperitoneal injection of both E. saphenum and P. gingivalis, indicating that E. saphenum may play just as important a role in periodontal diseases as P. gingivalis.

Germinated barley foodstuff feeding. A novel neutraceutical therapeutic strategy for ulcerative colitis.

A germinated barley foodstuff (GBF) contained glutamine-rich protein and the hemicellulose-rich fiber was made from brewer's spent grain by physical isolation (milling and sieving). Both in vivo and in vitro studies demonstrated that the fiber fraction of GBF supports maintenance of epithelial cell populations, facilitates epithelial repair, and suppresses epithelial nuclear factor kappa B-DNA binding activity through generating increased short-chain fatty acid (especially butyrate) production by luminal microflora which includes Bifidobacterium and Eubacterium, thereby preventing experimental colonic injury. The fiber fraction also modulates stool water content by its high water-holding capacity. The protein fraction which contains larger glutamine prevents experimental small bowel injury. Based on these observations, clinical studies were initiated in patients with mild to moderate active ulcerative colitis. The patients who had been unresponsive to or intolerant of standard treatment received 30 grams of GBF feeding daily in a nonrandomized, open-label fashion. At 4 weeks, this treatment resulted in a significant clinical and endoscopic improvement independent of disease extent. The improvement was associated with an increase in stool butyrate concentrations and in luminal Bifidobacterium and Eubacterium levels. After the end of GBF treatment the patients had an exacerbation of the disease. GBF was safe and well tolerated. These results indicate that GBF feeding is a potentially attractive treatment in patients with ulcerative colitis.

Characterisation of Eubacterium cell wall: peptidoglycan structure determines arthritogenicity.

OBJECTIVE: To elucidate factors involved in the arthritogenicity of bacterial cell walls. METHODS: For characterisation of an arthritogenic Eubacterium aerofaciens cell wall, peptidoglycan-polysaccharide (PG-PS) polymers were isolated by removing cell wall associated proteins (CWPs), PG and PS moieties were separated, and an attempt was made to de-O-acetylate PG-PS. The cell wall of E limosum was used as a non-arthritogenic control. The chemical composition of these cell wall preparations was analysed by gas chromatography-mass spectrometry. Also, their ability to resist lysozyme degradation and to sustain experimental chronic arthritis was tested. RESULTS: The observations made with the cell wall of E aerofaciens, an anaerobic habitant of the human intestine, were compared with those reported from a pathogenic Streptococcus, showing that in both strains a complex consisting of PG-PS is required for the induction of chronic arthritis. The PS moiety most probably protects PG from enzyme degradation, allowing prolonged tissue persistence and leading to the chronic synovial inflammation. CWPs attached to PG-PS are not necessary for this function. O-Acetylation of PG, which is required for arthritogenicity of the streptococcal cell wall, seems not to be present in the arthritogenic E aerofaciens PG or only occurs to a small degree; attempts to de-O-acylate the E aerofaciens cell wall did not affect its arthritogenicity or lysozyme resistance. CONCLUSION: The results obtained indicate that the source of bacterial cell wall plays no part in the chemical or structural requirements for PG to induce chronic cell wall arthritis in the rats; the chemical structure of the PG moiety is decisive.

The gingival immune response to periodontal pathogens in juvenile periodontitis.

A gingival explant culture system was utilized to evaluate the reactivity of local immunoglobulins produced by juvenile periodontitis tissue. Gingival explant culture supernatant fluids were screened, via a standardized dot-immunobinding assay, for antibodies reactive to: Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, Prevotella intermedia, Campylobacter rectus, Eikenella corrodens, Peptostreptococcus micros, Peptostreptococcus anaerobius, Capnocytophaga ochracea, Eubacterium nodatum and Fusobacterium nucleatum and one nonoral microorganism, Porphyromonas asaccharolytica. Of the 75 juvenile periodontitis supernatant fluids tested, the organisms that reacted with the highest numbers of supernatant fluids were E. nodatum (72%) and A. actinomycetemcomitans (49%). More juvenile periodontitis than healthy tissue samples showed supernatant fluid reactivity to P. intermedia, C. ochracea, E. nodatum and P. micros. No significant difference was observed between the juvenile periodontitis group supernatant fluids reactivity and the supernatant fluids of the other periodontal disease groups tested. Cluster analysis revealed the association, as determined by supernatant fluid reactivity, of P. micros and C. ochracea in the juvenile periodontitis group. The data from this investigation are consistent with a hypothesis of multiple possible etiologies of periodontal destruction in juvenile periodontitis and other forms of periodontal diseases.

Splenic granulomas in broiler chickens produced experimentally by inoculation with Eubacterium tortuosum.

Eubacterium tortuosum, a gram-positive anaerobic filamentous bacillus, was isolated from splenic and hepatic granulomas of a 56-day-old slaughtered chicken. This isolate was injected intravenously into two groups of 2-week-old broiler chickens, which were necropsied 19 days later. Five of 15 chickens injected with 5 x 10(6) colony-forming units of a 48-hour culture of E. tortuosum developed splenic granulomas typical of those seen in chickens at slaughter. No lesions were observed in chickens given 5 x 10(5) colony-forming units of E. tortuosum or in control chickens receiving phosphate-buffered saline solution. Attempts to reisolate E. tortuosum from experimentally infected chickens were unsuccessful; however, typical filamentous organisms were observed in splenic granulomas of all five affected chickens.

Significant recovery of nonsporulating anaerobic rods from clinical specimens.

Eighteen isolates of Bifidobacterium species, 99 of Eubacterium species, and 38 of anaerobic Lactobacillus species were recovered from 3,971 clinical specimens submitted to the anaerobic microbiology laboratory at the National Naval Medical Center over a period of 10 years (June 1978 to June 1988). Clinically significant infection was documented in association with 53 isolates recovered from 52 patients: 8 (44%) of the 18 Bifidobacterium isolates, 30 (30%) of the 99 Eubacterium isolates, and 15 (39%) of the 38 Lactobacillus isolates. The rest of the isolates were considered to be contaminants or to be of uncertain pathogenic significance. The significant infections that were documented mostly involved abdominal abscesses, obstetric and gynecologic sites, and wounds. Predisposing conditions (primarily prior surgery, immunodeficiency, malignancy, presence of a foreign body, or diabetes) were apparent in 7 (87.5%) of the 8 patients infected with Bifidobacterium species, in 23 (85%) of the 27 patients infected with Eubacterium species for whom clinical records were available, and in 8 (67%) of the 12 patients infected with Lactobacillus species for whom clinical records were available. Antimicrobial therapy was administered to 40 (85%) of the 47 patients for whom clinical records were available; such treatment was given in conjunction with surgical drainage or correction for 31 of these 47 patients (66%). No patient died of infection due to anaerobic, nonsporulating, gram-positive rods. These data illustrate that, although Bifidobacterium, Eubacterium, and Lactobacillus species are infrequently associated with infections, they occasionally do cause serious illness.

Influence of decontamination on induction of arthritis in Lewis rats by cell wall fragments of Eubacterium aerofaciens. Arthropathic properties of indigenous anaerobic bacteria.

Although the cause (or causes) of rheumatoid arthritis is unknown, many workers have suggested that microorganisms play a part. The intestinal flora in particular has been related to the development of joint inflammation. It has been shown previously that cell wall fragments of several anaerobic Gram positive intestinal bacteria of human origin are arthritogenic after a single intraperitoneal injection in Lewis rats. The part played by indigenous microflora in this model has now been studied by decontaminating Lewis rats before the injection of Eubacterium aerofaciens cell wall fragments. The pattern and severity of arthritis appeared to be comparable in decontaminated and control rats. The second goal of this work was to isolate arthritogenic bacteria from the autochthonous intestinal flora of rats. Only a limited number of bacteria showing a resemblance to arthritogenic strains from human intestinal flora (i.e. E aerofaciens and Bifidobacterium adolescentis) could be isolated. These strains did not induce chronic arthritis after intraperitoneal injection. This may explain why spontaneous arthritis did not develop in Lewis rats.

Arthritis by autoreactive T cell lines obtained from rats after injection of intestinal bacterial cell wall fragments.

T cell lines (B13, B19) were isolated from the lymph nodes of Lewis rats 12 days after an arthritogenic injection of cell wall fragments of Eubacterium aerofaciens (ECW), a major resident of the human intestinal flora. These cell wall fragments consist of peptidoglycan polysaccharide complexes (PPC). The cell lines that bear the helper phenotype were arthritogenic in knee or ankle joints upon intravenous injection into irradiated Lewis recipients. B13 was, however, not arthritogenic in irradiated F344 recipients that are largely RT1 identical. The arthritis induced in the knee joints of the irradiated Lewis rats was clearly shown by a 99mtechnetium-pertechnetate scanning technique and was confirmed histologically. In vitro the cell lines showed a proliferative response after stimulation with syngeneic spleen cells alone. The proliferation was significantly higher when bacterial PPC, isolated in soluble form from normal feces or ileostomy fluid were added. Recognition by B13 appeared to be MHC class II restricted. These results show that autoreactive T cell lines can be isolated from rats after injection of bacterial cell wall antigens and that these cell lines can be arthritogenic. This suggests a role for autoreactive T cells in the induction of bacterial cell wall arthritis and might give a clue for the arthritogenic properties of the normal human intestinal flora.

Reactivity of IgG from explant cultures of periapical lesions with implicated microorganisms.

The presence of IgG in periapical inflammatory lesions suggests that immune responses participate in the disease process. The purpose of this investigation was to study the reactivity of IgG from the supernatant fluids of explant cultures of periapical lesions with microorganisms implicated in infections of endodontic origin. Ninety periapical lesions that had been contiguous with the apex of a root were removed and maintained in explant cultures. A dot-enzyme-linked immunosorbent assay (dot-ELISA) was used to demonstrate the presence of IgG in the supernatant fluids of the explant cultures reactive with a panel of microorganisms associated with infections of endodontic origin. The percentages of reactivity by dot-ELISA follow: Bacteroides intermedius (84%), B. buccae (12%), Porphyromonas (Bacteroides) gingivalis (50%), P. endodontalis (58%), P. asaccharolyticus (17%), Peptostreptococcus micros (44%), P. anaerobius (26%), Eubacterium alactolyticum (34%), Fusobacterium nucleatum (14%), and Actinomyces israelii (6%). At least one of the three species of B. intermedius, P. gingivalis, or P. endodontalis tested gave a positive dot-ELISA with 89% of the supernatant fluids from explant cultures of periapical lesions. A lack of cross reactivity of IgG in supernatant fluids from explants of periapical lesions was demonstrated for the four strains of black-pigmented Bacteroides/Porphyromonas by dot-ELISA.

Pathogenic potential of Eubacterium yurii subspecies.

Several mechanisms that could contribute to the periodontopathogenic potential of Eubacterium yurii were investigated. All 18 strains examined produced RNAase and the metabolites H2S, indole and butyrate. Some strains produced phosphatase and DNAase. Methanol extracts of whole cells of E. yurii subspp. yurii and margaretiae stimulated bone resorption in vitro comparable to that produced by recognised periodontal pathogens. These results suggest that further studies should be performed to elucidate the role of E. yurii in periodontal disease.

Chronic arthritis induced in rats by cell wall fragments of Eubacterium species from the human intestinal flora.

To investigate arthritis-inducing properties of Eubacterium species, which are major residents of the human intestinal flora, cell wall fragments (CWF) of several Eubacterium strains were prepared and tested in an animal model. After a single intraperitoneal injection in the rat, CWF of E. aerofaciens, E. contortum, and E. lentum induced a chronic polyarthritis. E. limosum and E. tortuosum CWF induced an acute self-limiting joint inflammation, whereas E. rectale CWF failed to do so. The rhamnose contents of the isolated CWF were not related to their arthritis-inducing properties. Paradoxically, the sensitivity of CWF to lysozyme digestion, which is regarded as a parameter for the clearance of CWF in tissues, appeared to be positively correlated with the ability of Eubacterium CWF to induce chronic joint inflammation. Our findings show the diversity in arthritis-inducing properties among different species of the anaerobic genus Eubacterium and underline the importance of the anaerobic intestinal flora in the induction of joint inflammation.