Serum IgG titers against periodontal pathogens are associated with cerebral hemorrhage growth and 3-month outcome.

To assess the influence of periodontal disease on cerebral hemorrhage and its clinical course, we examined the association of the serum IgG titer of periodontal pathogens with hemorrhage growth and 3-month outcome. We consecutively enrolled 115 patients with acute cerebral hemorrhage (44 females, aged 71.3 ± 13.1 years) and used ELISA to evaluate the serum IgG titers of 9 periodontal pathogens: Porphyromonas gingivalis, Aggregatibacter (A.) actinomycetemcomitans, Prevotella intermedia, Prevotella nigrescens, Fusobacterium (F.) nucleatum, Treponema denticola, Tannerella forsythensis, Campylobacter rectus, and Eikenella corrodens. Significant hematoma growth was defined as an increase in the volume of >33% or an absolute increase in the volume of >12.5 mL. A poor outcome was defined as a 3 or higher on the modified Rankin Scale. We observed hemorrhage growth in 13 patients (11.3%). Multivariate analysis revealed that increased IgG titers of A. actinomycetemcomitans independently predicted the elevated hemorrhage growth (odds ratio 5.26, 95% confidence interval 1.52-18.25, p = 0.01). Notably, augmented IgG titers of F. nucleatum but not A. actinomycetemcomitans led to a poorer 3-month outcome (odds ratio 7.86, 95% confidence interval 1.08-57.08, p = 0.04). Thus, we demonstrate that elevated serum IgG titers of A. actinomycetemcomitans are an independent factor for predicting cerebral hemorrhage growth and that high serum IgG titers of F. nucleatum may predict a poor outcome in patients with this disease. Together, these novel data reveal how systemic periodontal pathogens may affect stroke patients, and, should, therefore, be taken into consideration in the management and treatment of these individuals.

Systemic proinflammation after Mycobacterium tuberculosis infection was correlated to the gut microbiome in HIV-uninfected humans.

BACKGROUND: The dysbiosis of gut microbiome and interaction with host immunity after Mycobacterium tuberculosis (MTB) infection are under investigation. We had found fatigue symptom concurrent with dysbiosis by decreasing the ratio of Firmicutes to Bacteroidetes (F/B ratio) in active tuberculosis (TB). The study aims to assess the inflammatory biomarkers and their interaction with gut microbiome in active TB and latent TB infection before starting anti-TB regimens. MATERIALS AND METHOD: Interleukin-1 beta (IL-1B), IL-4, IL-6, IL-10, CD3+, CD4+, CD8+ T cells and interferon-gamma (IFN-γ) releasing assay (IGRA) were measured in 25 active TB patients, 32 LTBI subjects and 23 healthy controls (HC). Gut microbiome profiles were obtained using 16S rRNA MiSeq sequencing method. RESULTS: The leucocytosis (7032 ± 387 cell/cum, P < 0.05), increase in IL-6 (229.7 ± 104 µg/dL, P < 0.05), and decrease in IL-4 (0.27 µg/dL ± 0.1, P < 0.05) were presented in active TB. The proportion of polymorphic neutrophil (PMN) in peripheral blood was positively related to the relative abundance of Bacteroidetes in LTBI and active TB (R2  = 0.23, P < 0.05). The F/B ratio was positively related to the detectable IL-1B in TB (R2  = 0.97, P < 0.01) and to the IL-4 in LTBI (R2  = 0.27, P < 0.05). In LTBI, the relative abundances of Coriobacteriaceae were positively related to the secretion of IFN-gamma against MTB-antigens more likely associated with of CD4+ T cell (R2  = 0.42, P < 0.05). CONCLUSION: In active TB, dysbiosis with higher relative abundances of Bacteroidetes in stool and low F/B ratio was related to systemic proinflammation. In LTBI, dose-response relationship between peripheral PMN and relative abundances of Bacteroidetes was remained but not leads to systemic inflammation.

Microbial dysbiosis associated with impaired intestinal Na+/H+ exchange accelerates and exacerbates colitis in ex-germ free mice.

Intestinal epithelial Na+/H+ exchange facilitated by the apical NHE3 (Slc9a3) is a highly regulated process inhibited by intestinal pathogens and in inflammatory bowel diseases. NHE3-/- mice develop spontaneous, bacterially mediated colitis, and IBD-like dysbiosis. Disruption of epithelial Na+/H+ exchange in IBD may thus represent a host response contributing to the altered gut microbial ecology, and may play a pivotal role in modulating the severity of inflammation in a microbiome-dependent manner. To test whether microbiome fostered in an NHE3-deficient environment is able to drive mucosal immune responses affecting the onset or severity of colitis, we performed a series of cohousing experiments and fecal microbiome transplants into germ-free Rag-deficient or IL-10-/- mice. We determined that in the settings where the microbiome of NHE3-deficient mice was stably engrafted in the recipient host, it was able accelerate the onset and amplify severity of experimental colitis. NHE3-deficiency was characterized by the reduction in pH-sensitive butyrate-producing Firmicutes families Lachnospiraceae and Ruminococcaceae (Clostridia clusters IV and XIVa), with an expansion of inflammation-associated Bacteroidaceae. We conclude that the microbiome fostered by impaired epithelial Na+/H+ exchange enhances the onset and severity of colitis through disruption of the gut microbial ecology.

Periodontal bacteria in human carotid atherothrombosis as a potential trigger for neutrophil activation.

OBJECTIVE: Epidemiological, biological and clinical links between periodontal and cardiovascular diseases are now well established. Several human studies have detected bacterial DNA corresponding to periodontal pathogens in cardiovascular samples. Intraplaque hemorrhage has been associated with a higher risk of atherosclerotic plaque rupture, potentially mediated by neutrophil activation. In this study, we hypothesized that plaque composition may be related to periodontal pathogens. METHODS: Carotid culprit plaque samples were collected from 157 patients. Macroscopic characterization was performed at the time of collection: presence of blood, lipid core, calcification and fibrosis. Markers of neutrophil activation released by carotid samples were quantified (myeloperoxidase or MPO, cell-free DNA and DNA-MPO complexes). PCR analysis using specific primers for Porphyromonas gingivalis, Aggregatibacter actinomycetemcommitans, Treponema denticola, Prevotella intermedia and Tannerella forsythia was used to detect DNA from periodontal pathogens in carotid tissues. In addition, bacterial lipopolysaccharide (LPS) and Immunoglobulins G against T. forsythia were quantified in atherosclerotic carotid conditioned medium. RESULTS: Intraplaque hemorrhage was present in 73/157 carotid samples and was associated with neutrophil activation, reflected by the release of MPO, cell-free DNA and MPO-DNA complexes. LPS levels were also linked to intraplaque hemorrhage but not with the neutrophil activation markers. Seventy-three percent of the carotid samples were positive for periodontal bacterial DNA. Furthermore, hemoglobin levels were associated with the detection of T. forsythia and neutrophil activation/inflammation markers. CONCLUSION: This study suggests a potential role of periodontal microorganisms, especially T. forsythia, in neutrophil activation within hemorrhagic atherosclerotic carotid plaques.

In vitro reconstruction of inflammatory reaction in human semen: effect on sperm DNA fragmentation.

The study was aimed at evaluating an in vitro induction of DNA damage in three sperm subpopulations exposed to selected inflammatory mediators, such as leukocytes, two combinations of pro-inflammatory cytokines (interleukin [IL]-6 + IL-8 and IL-12 + IL-18) and two bacterial strains (Escherichia coli and Bacteroides ureolyticus). Semen samples from normozoospermic volunteers were differentiated by swim-up (swim-up fraction) and Percoll gradient procedures (90% and 47% Percoll fractions). Leukocytes were isolated from the whole heparinized blood using the density gradient centrifugation technique. DNA fragmentation in sperm fractions was evaluated using flow cytometry with TUNEL labeling and Comet assay. Out of the inflammatory factors tested, bacteria were found to have a greatest toxic effect on sperm DNA, especially in fractions isolated by Percoll gradient, compared with untreated cells (P < 0.05). The results indicate that inflammatory mediators can be a direct cause of DNA fragmentation in ejaculated spermatozoa, which can ultimately lead to limited fertilizing abilities of the germ cells. In contrast to the swim-up technique, the selection of spermatozoa by gradient procedures increases the vulnerability of mature spermatozoa to the harmful effects of infectious agents on DNA integrity. This observation may have some meaning for recommendations concerning laboratory techniques used in assisted reproductive therapy.

Characterizing the S-layer structure and anti-S-layer antibody recognition on intact Tannerella forsythia cells by scanning probe microscopy and small angle X-ray scattering.

Tannerella forsythia is among the most potent triggers of periodontal diseases, and approaches to understand underlying mechanisms are currently intensively pursued. A ~22-nm-thick, 2D crystalline surface (S-) layer that completely covers Tannerella forsythia cells is crucially involved in the bacterium-host cross-talk. The S-layer is composed of two intercalating glycoproteins (TfsA-GP, TfsB-GP) that are aligned into a periodic lattice. To characterize this unique S-layer structure at the nanometer scale directly on intact T. forsythia cells, three complementary methods, i.e., small-angle X-ray scattering (SAXS), atomic force microscopy (AFM), and single-molecular force spectroscopy (SMFS), were applied. SAXS served as a difference method using signals from wild-type and S-layer-deficient cells for data evaluation, revealing two possible models for the assembly of the glycoproteins. Direct high-resolution imaging of the outer surface of T. forsythia wild-type cells by AFM revealed a p4 structure with a lattice constant of ~9.0 nm. In contrast, on mutant cells, no periodic lattice could be visualized. Additionally, SMFS was used to probe specific interaction forces between an anti-TfsA antibody coupled to the AFM tip and the S-layer as present on T. forsythia wild-type and mutant cells, displaying TfsA-GP alone. Unbinding forces between the antibody and wild-type cells were greater than with mutant cells. This indicated that the TfsA-GP is not so strongly attached to the mutant cell surface when the co-assembling TfsB-GP is missing. Altogether, the data gained from SAXS, AFM, and SMFS confirm the current model of the S-layer architecture with two intercalating S-layer glycoproteins and TfsA-GP being mainly outwardly oriented.

Persistently high levels of periodontal pathogens associated with preterm pregnancy outcome.

BACKGROUND: Few studies examining the association between periodontal diseases and preterm birth have explored the underlying microbial and antibody responses associated with oral infection. METHODS: A nested case-control study was performed using data from a recent interventional trial following the delayed-treatment control group of 31 subjects with periodontal diseases. The levels of eight oral bacteria and the maternal immunoglobulin G (IgG) responses in serum to these bacteria were measured at antepartum and postpartum visits to determine the relationship to cases (preterm delivery <37 weeks' gestation) and controls (term delivery). RESULTS: Antepartum, the levels of periodontal pathogens tended to be higher in the preterm (case group) deliveries compared to the term deliveries (control group). Maternal anti-Porphyromonas gingivalis IgG was significantly lower in the preterm group compared to the term group (P = 0.028). Postpartum, levels of P. gingivalis, Tannerella forsythia, Prevotella intermedia, and Prevotella nigrescens were statistically significantly higher in preterm births compared to term deliveries, adjusting for baseline levels. The joint effects of red and orange microbial clusters were significantly higher in the preterm group compared to the term group. CONCLUSIONS: High levels of periodontal pathogens and low maternal IgG antibody response to periodontal bacteria during pregnancy are associated with an increased risk for preterm delivery. Further studies elucidating the role of the microbial load and maternal immune response as related to pregnancy outcome seem merited.

Inflammatory responses of a macrophage/epithelial cell co-culture model to mono and mixed infections with Porphyromonas gingivalis, Treponema denticola, and Tannerella forsythia.

Accumulated evidence points to Porphyromonas gingivalis, Treponema denticola, and Tannerella forsythia as three major etiologic agents of chronic periodontitis. Epithelial cells and macrophages play a major role in the host response to periodontopathogens, and the secretion of inflammatory mediators and matrix metalloproteinases (MMPs) by these host cells is believed to contribute to periodontal tissue destruction. The aim of this study was to investigate the inflammatory response of a macrophage/epithelial cell co-culture model following mono or mixed infections with the above three periodontopathogens. An in vitro co-culture model composed of epithelial-like transformed cells (HeLa cell line) and macrophage-like cells (phorbol myristic acid-differentiated U937 monocytic cell line) was challenged with whole cells or lipopolysaccharides (LPS) of P. gingivalis, T. denticola, and T. forsythia, individually and in combination. Following stimulation, the production of interleukin-1 beta (IL-1beta), IL-6, IL-8, tumor necrosis factor alpha (TNF-alpha), regulated on activation normal T cell expressed and secreted (RANTES), prostaglandin E2 (PGE2), and MMP-9 were quantified by enzyme-linked immunoassays. We observed that mono or mixed infections of the co-culture model induced the secretion of IL-1beta, IL-6, IL-8, PGE2, and MMP-9. P. gingivalis and T. forsythia induced an increase in RANTES secretion, whereas T. denticola alone or in combination resulted in a significant decrease in RANTES levels. All LPS challenges induced an increase in chemokine, MMP-9, and PGE2 production. No synergistic effect on the production of cytokines, chemokines, PGE2, and MMP-9 was observed for any of the bacterial or LPS mixtures tested. This study supports the view that P. gingivalis, T. denticola, and T. forsythia may induce high levels of pro-inflammatory mediators and MMP-9 in periodontal lesions, thus contributing to the progression of periodontitis.

Structure of the polysaccharide chain of the lipopolysaccharide from Flexibacter maritimus.

Flexibacter maritimus, a Gram-negative bacterium, is a fish pathogen responsible for disease in finfish species and a cause of cutaneous erosion disease in sea-caged salmonids. For the development of serology based diagnostics, protective vaccines, and a study of pathogenesis, the structural analysis of the lipopolysaccharide (LPS) produced by the bacterium has been undertaken. We now report that an acidic O-specific polysaccharide, obtained by mild acid degradation of the F. maritimus LPS was found to be composed of a disaccharide repeating unit built of 2-acetamido-3-O-acetyl-4-[(S)-2-hydroxyglutar-5-ylamido]-2,4,6-trideoxy-beta-glucose and 5-acetamido-7-[(S)-3-hydroxybutyramido]-8-amino-3,5,7,8,9-pentadeoxynonulopyranosonic acid (Sug) having the structure: The configuration of the C-2-C-7 fragment of the latter monosaccharide (B) was assigned beta-manno; however, the configuration at C-8 could not be established. NMR data indicate that the two monosaccharides have opposite absolute configurations. The repeating unit includes a linkage via a (S)-2-hydroxyglutaric acid residue, reported here for the first time as a component of a bacterial polysaccharide. The LPS was also found to contain a minor amount of a disaccharide beta-Sug-(2-3)-l-Rha, isolated from the products of the acidic methanolysis of the LPS.

Granulocyte-macrophage colony-stimulating factor amplification of interleukin-1beta and tumor necrosis factor alpha production in THP-1 human monocytic cells stimulated with lipopolysaccharide of oral microorganisms.

Cytokines, including granulocyte-macrophage colony-stimulating factor (GM-CSF), are used to assist in bone marrow recovery during cancer chemotherapy. Interleukin-1beta (IL-1beta) and tumor necrosis factor alpha (TNF-alpha) play important roles in inflammatory processes, including exacerbation of periodontal diseases, one of the most common complications in patients who undergo this therapy. A human monocyte cell line (THP-1) was utilized to investigate IL-1beta and TNF-alpha production following GM-CSF supplementation with lipopolysaccharide (LPS) from two oral microorganisms, Porphyromonas gingivalis and Fusobacterium nucleatum. LPS of P. gingivalis or F. nucleatum was prepared by a phenol-water extraction method and characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and determination of total protein and endotoxin contents. Resting THP-1 cells were treated with LPS of P. gingivalis or F. nucleatum and/or GM-CSF (50 IU/ml) by using different concentrations for various time periods. Production of IL-1beta and TNF-alpha in THP-1 cells was measured by solid-phase enzyme-linked immunosorbent assay. Reverse transcription (RT)-PCR was used to evaluate the gene expression of resting and treated THP-1 cells. IL-1beta was not detected in untreated THP-1 cells. IL-1beta production was, however, stimulated sharply at 4 h. GM-CSF amplified IL-1beta production in THP-1 cells treated with LPS from both oral anaerobes. No IL-1beta-specific mRNA transcript was detected in untreated THP-1 cells. However, IL-1beta mRNA was detected by RT-PCR 2 h after stimulation of THP-1 cells with LPS from both organisms. GM-CSF did not shorten the IL-1beta transcriptional activation time. GM-CSF plus F. nucleatum or P. gingivalis LPS activated THP-1 cells to produce a 1.6-fold increase in TNF-alpha production at 4 h over LPS stimulation alone. These investigations with the in vitro THP-1 model indicate that there may be an increase in the cellular immune response to oral endotoxin following GM-CSF therapy, as evidenced by production of the tissue-reactive cytokines IL-1beta and TNF-alpha.

Molecular and immunological characterization of a 64-kDa protein of Actinobacillus actinomycetemcomitans.

The 64-kDa protein to which about half the sera from patients with localized juvenile periodontitis and rapidly progressive periodontitis reacted strongly was purified from Actinobacillus actinomycetemcomitans Y4. Determination of the N-terminal sequence of the protein revealed that it was a GroEL-like protein. The DNA fragment containing the groEL gene of A. actinomycetemcomitans was amplified by polymerase chain reaction, and the groESL operon was cloned by using colony hybridization with the amplified fragment from A. actinomycetemcomitans chromosomal DNA. Sequence analysis revealed that structures of the operon and its products were typical in gram-negative bacteria. Rabbit polyclonal antibodies to the 64-kDa protein cross-reacted with approximately 65-kDa proteins of Haemophilus aphrophilus, Haemophilus influenzae, Haemophilus paraphrophilus, Escherichia coli and Eikenella corrodens but not with any cellular proteins of Porphyromonas gingivalis, Prevotella intermedia and Fusobacterium nucleatum. It is possible that antibodies reactive to the 64-kDa protein in periodontitis patients are induced by the cross-reactivity with the hsp60 proteins of other bacteria.

Progress in the identification of nonmotile Bacteroidaceae from dental plaque.

Over the last decade, biochemical and chemical analyses have been used widely to study the intrageneric structure of Bacteroidaceae. New chromogenic substrates (e.g., naphthylamide-linked compounds) and fluorogenic substrates (e.g., 4-methylumbelliferyl or 7-amido-4-methyl-coumarin compounds) can be used to identify certain species within a few hours under aerobic conditions. Clarification of the taxonomy of many oral anaerobic species that are now considered important putative periodontal pathogens has permitted the development of panels of both polyclonal and monoclonal antibodies for their detection. Similarly, both DNA and RNA gene probes, derived through nucleic acid sequence analysis, have been constructed for several species; many such probes are now commercially available. In the long term, the application of these techniques will lead to a better understanding of the distribution, transmission, and ecological and clinical importance of many species that have hitherto remained poorly characterized.

A rapid method for preparation of rough and smooth lipopolysaccharide from Bacteroides, Porphyromonas and Prevotella.

We describe a new method for lipopolysaccharide (LPS) preparation by water extraction at 100 degrees C and subsequent digestion with proteinase K. The crude LPS could be reliably used for immunoblotting since it retained a high level of antigenicity, and was free of SDS and proteinase K, both of which can cause problems. Two monoclonal antibodies which failed to react with LPS prepared by two conventional methods reacted well with our preparation. We used the new method to prepare LPS from 44 strains of bacteria formerly classified as Bacteroides, some of which have been reclassified as Porphyromonas or Prevotella. In general, yields were good, and electrophoretic profiles obtained with SDS-PAGE and silver staining enabled strains to be rated rough, semi-rough, or smooth.

Virulence of Wolinella recta in a murine abscess model.

The virulence of Wolinella recta isolates was studied in an experimental animal model by using monoinfection of BALB/c mice. Infection with clinical isolates of W. recta 576 and W. recta 234 induced dry, flat, depressed gangrenous necrotic skin lesions, whereas W. recta ATCC 33238 failed to induce a similar lesion. Histological examination of the skin lesion 72 h postinfection revealed coagulation necrosis of the epidermis, subcutis and cutaneous truncus muscle, with marked exudation of serum proteins and neutrophils. Virulence-modulating agents such as dexamethasone, galactosamine, hydrazine sulfate, and dextran microcarrier beads were used in conjunction with W. recta infection. Dexamethasone, hydrazine sulfate, and dextran beads enhanced the infectivity and pathogenicity of W. recta for lesion formation and tissue destruction compared with what was found in untreated control mice. Galactosamine sensitization enhanced the virulence potential of W. recta to such an extent that a lethal outcome was observed. Laboratory passage of clinical isolates demonstrated a decreased virulence in high-passage strains, which correlated with the minimal virulence observed in the extensively passaged W. recta ATCC 33238. Serum immunoglobulin G (IgG) and IgM responses were detected in the serum of infected animals, and cross-reacting antibody indicated variation in the antigenic makeup of various W. recta strains. Enhanced IgG antibody responses were observed following the secondary challenge. Mice with acquired antibody response to initial infection remained susceptible to lesion formation with subsequent challenge, but the size of the lesion was significantly reduced, indicating partial protection. Serum IgG and IgM antibody levels were significantly increased by active immunization when compared with levels in mice which had recovered from infection. The immunization significantly decreased the lesion size; however, even these high levels of antibody failed to abrogate the lesion induction.

Monoclonal antibodies for the detection of 'periodontopathic' bacteria.

The development of indirect immunofluorescence assays using MAbs as specific probes has made it possible to detect a variety of suspected periodontal pathogens in subgingival plaque, with exquisite sensitivity. The studies demonstrate high reproducibility of the results if different MAbs are used to assess the same bacterial species. Bacteroides forsythus, Bact. gingivalis, Bact. intermedius and Wolinella recta, but not Actinobacillus actinomycetemcomitans were found in sites of adult periodontitis with high prevalence but quite distinct degrees of colonization. The colonization levels of Bact. forsythus and Bact. gingivalis were significantly associated with the probing depth of the lesions. The investigations demonstrate the usefulness of serological analysis of plaque and indicate that several of the organisms implicated in the aetiology of periodontal diseases may be tolerated in high numbers in subgingival lesions, without periodontal destruction.

Production and characterization of monoclonal antibodies against Bacteroides forsythus and Wolinella recta.

Hybrid cell lines producing monoclonal antibodies against Bacteroides forsythus or Wolinella recta were generated by fusion of SP2/0 or FO myeloma cells with splenocytes from Balb/c mice immunized with formalinized cells of B. forsythus FDC 331 or W. recta D13a-g, respectively. Enzyme-linked immunosorbent assays and indirect immunofluorescence tests were used to analyze the distribution of the recognized antigens on a panel of 70 strains representing 35 taxa, most of which are members of the oral flora. All monoclonal antibodies--eight against B. forsythus and six against W. recta--proved specific for the immunizing species. Four of the monoclonal antibodies against W. recta recognized antigens expressed by only some of the tested W. recta strains, thus confirming the earlier noted antigenic heterogeneity of this species. Antibody binding patterns consistent with those previously described or distinct for new serogroups could not, however, be observed. Four of the eight anti-B. forsythus monoclonal antibodies bound to only two of the three tested B. forsythus strains. All the remaining monoclonal antibodies detected every strain tested of the respective species against which they were raised. Preliminary results indicated that several of these antibodies should be very useful for the direct identification and quantification of these organisms in subgingival plaque.

Differentiation of ruminal bacterial species by enzyme-linked immunosorbent assay using egg yolk antibodies from immunized chicken hens.

Cross-reactivity among four species of ruminal bacteria was examined by using egg yolk antibodies from immunized Leghorn laying hens and an enzyme-linked-immunosorbent assay. The effects of the four species on the hens were compared on various days postimmunization. Hens injected with the same bacterial species had similar apparent antibody levels over the entire postimmunization period, but only Bacteroides ruminicola B1(4) and Selenomonas ruminantium D antigens elicited early increases in apparent antibody levels during weeks 2 and 3. Antibody cross-reactivity was greatly reduced by week 2, except for antibodies against Streptococcus bovis JB1.

Role of encapsulated anaerobic bacteria in synergistic infections.

The effect of encapsulation on the virulence, survival, and protection of anaerobic bacteria from phagocytosis is reviewed. Support for the importance of encapsulated Bacteroides sp. and anaerobic and facultative Gram-positive cocci (AFGPC) was provided by their higher recovery rate in oropharyngeal infections, compared to their number in the normal oral flora. Studies of the pathogenicity of anaerobic bacteria of the Bacteroides, Fusobacterium, and Clostridium genera and AFGPC are also presented. The organisms were inoculated into mice and their ability to induce subcutaneous abscesses was determined. Encapsulated Bacteroides, Fusobacteria, and AFGPC generally induced abscesses, whereas unencapsulated organisms did not. However, many of the strains that had only a minimal number of encapsulated organisms (less than 1%) survived in the abscess, and became heavily encapsulated when inoculated with other viable or nonviable encapsulated bacteria. These strains were thereafter able to induce abscesses when injected alone. Encapsulated Bacteroides sp. and anaerobic cocci induced bacteremia and translocation, and increased the mortality of the infected animals more often than did the unencapsulated form of the same strains. The relative importance of encapsulated anaerobes in relation to their aerobic and facultative counterparts in mixed infection was studied, using selective antimicrobial therapy and quantitative cultures of abscesses induced in mice. With few exceptions, possession of a capsule made Bacteroides sp. more important than their aerobic counterparts. Synergistic potentials were seen between encapsulated Bacteroides sp. and all tested aerobic bacteria and most AFGPC, and between most AFGPC and Pseudomonas aeruginosa or Staphylococcus aureus. These studies demonstrated the importance of encapsulated anaerobes in mixed infections.

Reaction of human sera from juvenile periodontitis, rapidly progressive periodontitis, and adult periodontitis patients with selected periodontopathogens.

The levels of serum antibody reactive to selected periodontopathogens were determined in 182 clinically characterized patients: 35 healthy control, 50 juvenile periodontitis, 42 adult periodontitis and 55 rapidly progressive periodontitis. Reactive antibody levels were determined using an enzyme-linked immunosorbent assay with whole cell preparations of Bacteroides gingivalis, Capnocytophaga (Bacteroides) ochraceus, Fusobacterium nucleatum and Actinobacillus actinomycetemcomitans (Y-4) serving as antigens. Increased reactivity to B. gingivalis and F. nucleatum was observed in all three disease groups studied while antibody reactive to A. actinomycetemcomitans was increased in juvenile and rapidly progressive periodontitis. Antibody levels reactive to C. ochraceus in healthy subjects did not differ from those observed in any disease patient groups. Possible implications in the etiology and progression of the diseases coupled with environmental changes which occur in the econiche of the periodontal pocket are described.