Direct hemagglutination technique for differentiating Bacteroides asaccharolyticus oral strains from nonoral strains.

A simple and economical method for differentiating Bacteroides asaccharolyticus of oral sources from nonoral sources is described. The present data indicate that oral strains of B. asaccharolyticus strongly agglutinate sheep erythrocytes, whereas isolates from various nonoral sites typically are devoid of hemagglutination activity. The direct hemagglutination test may aid in determining the source of B. asaccharolyticus present in an infection, and thus the procedure has potential value as a means of biotyping.     

Evaluation of Fluoretec-M for detection of oral strains of Bacteroides asaccharolyticus and Bacteroides melaninogenicus.

Fluoretec-M is a polyvalent conjugate used in direct fluorescent-antibody staining for identification of the Bacteroides asaccharolyticus-Bacteroides melaninogenicus group. The Fluoretec-M reagent detected all oral and nonoral test strains of B. melaninogaenicus subsp. intermedius, all test strains of B. melaninogenicus subsp. melaninogenicus, and the nonoral strains of B. asaccharolyticus. However, the Fluoretec-M polyvalent reagent and the monovalent conjugates which constitute Fluoretec-M did not detect the oral strains B. asaccharolyticus. The use of Fluoretec-M can therefore generate false-negative results in studies of specimens from oral cavity and from nonoral sites in which an infection with B. asacacharolyticus of oral origin may have taken place. It is suggested that antibodies reactive with the oral antigenic type of B. asaccharolyticus be included in the preparative procedure of the Fluoretec-M reagent.     

Production of phenylacetic acid by strains of Bacteroides asaccharolyticus and Bacteroides gingivalis (sp. nov.).

Strains of Bacteroides asaccharolyticus and Bacteroides melaninogenicus subspecies isolated from human and animal sources were examined for the production of phenylacetic acid. B. asaccharolyticus strains isolated from sites in humans and monkeys always produced phenylacetic acid. B. asaccharolyticus strains isolated from human nonoral sites consistently failed to produce this product. This metabolic difference correlates with the genetic dichotomy recently found to exist between oral and nonoral B. asaccharolyticus strains.     

Cytotoxic activity of Bacteroides gingivalis and Bacteroides asaccharolyticus

Culture filtrates of all eight strains of Bacteroides gingivalis and all five strains of B. asaccharolyticus were toxic for Vero cells. Cytotoxicity was in general greater with material from cultures of B. gingivalis than from B. asaccharolyticus but none of the culture filtrates from eight strains of B. melaninogenicus showed activity in this test. The toxic material was released during prolonged incubation and more detailed study of preparations from one strain indicated that it had a molecular weight of less than 3500 and was heat stable.     

Serum antibodies to oral Bacteroides asaccharolyticus (Bacteroides gingivalis): relationship to age and periondontal disease.

An enzyme-linked immunosorbent assay microplate method was used for measuring levels of antibody specific for the oral serotype of Bacteroides asaccharolyticus (Bacteroides gingivalis) in serum samples obtained from umbilical cords, infants, children, periodontally normal adults, and edentulous adults. Serum from patients with various periodontal diseases, including adult periodontitis, localized juvenile periodontitis, generalized juvenile periodontitis, post-localized juvenile periodontitis, and acute necrotizing ulcerative gingivitis, were also studied. A positive correlation between increase in age and increase in both prevalence and level of specific antibody in the G, A, and M classes of immunoglobulins was observed. This indicates that antibodies reactive with oral B. asaccharolyticus found in up to 84% of normal adults are natural antibodies, presumably with a protective role. Among the patient groups, those with adult periodontitis were found to have levels of immunoglobulin G antibodies to oral B. asaccharolyticus that were five times higher than the antibody levels found in control subjects. The levels of IgG antibodies to this organism in the other patient groups were comparable to the levels found in the control group. However, 50% of the individuals in the generalized juvenile periodontitis group had high levels of immunoglobulin G antibodies to B. asaccharolyticus, suggesting heterogeneity with respect to immune response in these patients. These results indicate that antibodies to oral B. asaccharolyticus (B. gingivalis) occur at low levels in most normal children and adults and that the rise in titer of the specific antibodies of each major class of immunoglobulins parallels the ontogenic change in serum levels of that isotype. In contrast, there is a marked increase in titer of immunoglobulin G antibodies to oral B. asaccharolyticus in the group of patients with adult periodontitis and in patients with the generalized form of juvenile periodontitis.     

Rapid characterization of oral and nonoral pigmented Bacteroides species with the ATB Anaerobes ID system.

The ATB Anaerobes ID system (API SYSTEM, La Balme Les Grottes, France) was evaluated for its ability to differentiate between species of the pigmented Bacteroides group. This identification system is based on the degradation of chromogenic substrates in combination with sugar fermentation reactions. The results showed that the ATB system can be useful for differentiation between the 10 pigmented Bacteroides species. However, additional tests may be necessary.     

Characteristics of Bacteroides asaccharolyticus from dental plaques of beagle dogs.

Gram-negative, non-saccharolytic, brown- or black-pigment-forming, nonmotile anaerboic coccobacilli, capable of decomposing hydrogen peroxide and identified as Bacteroides asaccharolyticus (B. melaninogenicus subsp. asaccharolyticus), were isolated from the supra- and subgingival plaques of beagle dogs with gingivitis or periodontitis. The organisms remained viable for many hours in an aerobic atmosphere as evidenced by their ability to grow subsequently in an anaerobic environment. They also grew well on agar media that were not reduced before use. Although blood was required for pigmentation of colonies, organisms grew on media that lacked hemin, menadione, blood, or reducing compounds. Increased oxygen tolerance, catalase activity, and different nutritional requirements differentiate these organisms from strains of B. asaccharolyticus isolated from humans.     

Oral implantation of Bacteroides asaccharolyticus and Eikenella corrodens in conventional hamsters.

Oral implantation of Bacteroides asaccharolyticus 381-R' and Eikenella corrodens 1073S-R, which are highly resistant to streptomycin, was examined in conventional hamsters. The hamsters' first molars were ligatured with cotton threads preimmersed in bacterial suspensions. Bacterial inoculation was performed daily for 1 week, followed by a single weekly inoculation for 7 more weeks. Hamsters were fed Keyes' diet no. 2000 or ordinary powdered diet. Bacterial recovery, gingival histological changes, and immunological response were checked 2, 5, and 8 weeks after the daily serial inoculation. B. asaccharolyticus 381-R' was recovered from all hamsters during the experiment (6.7 X 10(6) to 6.3 X 10(8) colony-forming units). E. corrodens 10735-R was recovered from the hamsters fed Keyes' diet no. 2000 throughout the experimental period (2.4 X 10(5) to 1.2 X 10(6) colony-forming units), but not in the group fed the ordinary powdered diet. The histological changes in gingival tissues at the second week showed no differences among the control group, the B. asaccharolyticus-inoculated group, and the E. corrodens-inoculated group. However, by the eighth week, the inflammation persisted only in the bacteria-inoculated groups, especially in the B. asaccharolyticus-inoculated group. A relatively increased serum antibody titer was also observed in the B. asaccharolyticus-inoculated group, but not in the E. corrodens-inoculated group. These findings indicate that B. asaccharolyticus 381-R' and E. corrodens 1073S-R can be implanted in hamsters' oral cavity with the aid of cotton thread ligature. It was also suggested that B. asaccharolyticus may have some pathogenic role in the destruction of periodontal tissue in hamsters.     

Immunochemical evidence for multiple serotypes of Bacteroides fragilis.

An immunochemical comparison of outer membrane antigens obtained from five select and biochemically defined strains indicated that there are several serotypes of Bacteroides fragilis. Each strain was serologically defined by individual or by combinations of determinant groups composed of carbohydrates in the form of polysaccharides or glycoproteins. The carbohydrate constituents were tentatively identified as glucose, galactose, fucose, rhamnose, glucosamine, galactosamine, and traces of mannose. Strains were observed to have minor qualitative and major quantitative variations in carbohydrate composition.     

Effects of Bacteroides asaccharolyticus cells and B. fragilis surface components on serum opsonisation and phagocytosis

The effect of Bacteroides asaccharolyticus on the opsonisation and phagocytosis of gram-positive and gram-negative bacteria by human polymorphonuclear leukocytes (PMNL) was investigated. Uptake of most isolates of staphylococci, streptococci and clostridia by PMNL after opsonisation with serum treated with B. asaccharolyticus was largely unimpaired. The same treatment of serum before opsonisation of isolates of Pseudomonas, Enterobacter, Klebsiella and Gardnerella resulted in the uptake by PMNL varying with individual isolates; a large reduction occurred with some and none with others. Treatment of serum with B. fragilis lipopolysaccharide before opsonisation of Proteus mirabilis produced a marked reduction in uptake, whereas treatment with B. fragilis capsular polysaccharide had little effect.     

Immunochemical characterization of two surface polysaccharides of Bacteroides fragilis.

Immunochemical analysis of the capsular polysaccharide from Bacteroides fragilis NCTC 9343 revealed a novel structure composed of two distinct polysaccharides. Immunoelectrophoresis of an extract of purified surface polysaccharide from fermenter-grown organisms showed a complex precipitin profile with varying anodal mobility. DEAE-Sephacel anion-exchange chromatography of the polysaccharide extract failed to separate the majority of this aggregate. Disaggregation of this complex was accomplished by very mild acid treatment; purification was achieved by DEAE-Sephacel anion-exchange chromatography. Polysaccharide A had a neutral charge at pH 7.3, a net negative charge at pH 8.6, and an average Mr = 110,000; chemical analysis showed it to contain galactose, galactosamine, and an unidentified amino sugar. Polysaccharide B eluted from the anion-exchange column with increased salt concentration; it had a net negative charge and an average Mr = 200,000, and contained fucose, galactose, quinovosamine, galacturonic acid, and glucosamine. Neither of these polysaccharides contained detectable 3-deoxy-D-manno-octolusonic acid, and both were recognized as distinct antigens on the basis of their reactivity with monoclonal antibodies CE3 and F10, which reacted with the complex before acid treatment. These data indicate that the capsule of B. fragilis NCTC 9343 comprises two discrete, surface-exposed polysaccharides with differing physiochemical properties that are distinct from the lipopolysaccharide of this organism. The finding of two surface polysaccharides has not been described for other bacteria pathogenic to humans.     

Serological studies of oral Bacteroides intermedius.

Bacteroides intermedius is a gram negative, anaerobic microorganism associated with certain forms of human periodontal disease, including adult periodontitis and acute necrotizing ulcerative gingivitis. Previous studies have indicated the presence of two DNA homology groups which could be distinguished by analysis of protein patterns on polyacrylamide gel electrophoresis, as well as at least two serogroups within B. intermedius. The present study examined the serology of B. intermedius and determined the distribution of B. intermedius serogroups in clinical isolates and patient plaque samples. Serological reactions with unabsorbed rabbit antisera and antisera immunoabsorbed with B. intermedius strains demonstrated a previously unreported antigenic group within B. intermedius, serogroup C, in both immunodiffusion and immunofluorescence assays. Of 79 B. intermedius isolates from 68 subjects examined with specific antisera, 55% of the isolates and 52% of the subjects were categorized in serogroup C, 40% of the isolates and 46% of the subjects were in serogroup B, and 5% of the isolates and 6% of the subjects were in serogroup A. In 31 samples of subgingival dental plaque from adolescents known to harbor B. intermedius, 81% demonstrated serogroup B, 16% had serogroup A, and 3% had serogroup C.     

Enzymatic characterization of some oral and nonoral gram-negative bacteria with the API ZYM system.

The API ZYM system (Analytab Products, Plainview, N.Y.), containing 19 chromogenic substrates, was utilized semiquantitatively to detect extracellular acid and alkaline phosphatases, aminopeptidases, proteases, esterase-lipase, phosphoamidase, and glycosidases in 128 oral and nonoral isolates of black-pigmented Bacteroides, Actinobacillus, Haemophilus aphrophilus, Capnocytophaga, Fusobacterium nucleatum, Wolinella recta, and Veillonella parvula. In the black-pigmented Bacteroides group of organisms, a strong trypsin reaction was present in Bacteroides gingivalis (oral species) but not in Bacteroides asaccharolyticus (nonoral species). Bacteroides melaninogenicus subsp. melaninogenicus, in contrast to Bacteroides melaninogenicus subsp. intermedius, exhibited strong N-acetyl-beta-glucosaminidase activity. H. aphrophilus produced beta-galactosidase and alpha-glucosidase, but the closely related Actinobacillus actinomycetemcomitans did not. Capnocytophaga was distinct with respect to strong aminopeptidase reactions. This study showed that a wide range of enzymes which have the potential of causing tissue injury and inflammation can be elaborated from major oral gram-negative species. Also, the API ZYM system appears to be a valuable adjunct to traditional biochemical testing in identifying oral gram-negative species.     

Immunochemical identification and preliminary characterization of a nonfimbrial hemagglutinating adhesin of Bacteroides gingivalis.

A cell-bound hemagglutinating adhesin (HA-Ag2) of Bacteroides gingivalis was identified by crossed immunoaffinity electrophoresis as one of the common antigens of the species. A polyclonal antiserum with a restricted specificity for HA-Ag2 was produced by immunizing with the relevant immunoprecipitate excised from crossed-immunoelectrophoresis gels. The immunoglobulin G fraction of this monospecific antiserum inhibited hemagglutination. The antiserum was used against a cell surface extract of B. gingivalis in immunoblotting experiments, and we detected two antigens with apparent molecular masses of 33 and 38 kilodaltons in B. gingivalis ATCC 33277 and W83. Monoclonal antibody, C1.17, produced in another laboratory against B. gingivalis 381 and characterized as showing reactivity with a hemagglutinin of this strain (Y. Naito, K. Okuda, T. Kato, and I. Takazoe, Infect. Immun. 50:231-235, 1985), was also used to produce immunoblots of extracts of strains ATCC 33277 and W83. The apparent molecular masses of the major polypeptides recognized by monoclonal C1.17 in the immunoblots were the same as those detected by the polyclonal monospecific antiserum, i.e., 33 and 38 kilodaltons. Significantly, none of the polypeptides identified in this study corresponded to the polypeptide appearing in the 41- to 43-kilodalton region and identified by Yoshimura and co-workers (F. Yoshimura, K. Takahashi, N. Yoshinobu, and T. Suzuki, J. Bacteriol. 160:949-957, 1984) as the fimbrial protein characteristic of the species. Enzyme-linked immunosorbent assay inhibition experiments with the monospecific antiserum indicated that the cell surface extracts from strains ATCC 33277 and W83 were strong inhibitors, whereas the fimbria-enriched preparations from both strains failed to inhibit binding of antibodies to the cell surface antigens. As a whole, our study indicates that a nonfimbrial surface protein complex demonstrating erythrocyte-binding capacity, HA-Ag2, is common to three strains of B. gingivalis and is composed of at least two associated polypeptides with apparent molecular masses of 33 and 38 kilodaltons which share at least one antigenic determinant.     

Bacteroides fragilis strains express multiple capsular polysaccharides.

Previous studies by our group have demonstrated that the capsule of Bacteroides fragilis type strain NCTC 9343 consists of two chemically distinct polysaccharides, designated PS A and PS B. These polysaccharides can be isolated as an aggregate from the surface of the organism and give a complex multiprecipitin profile when they are reacted with homologous antiserum in an immunoelectrophoresis assay. Following structural analysis of PS A and PS B, we have determined that the complex precipitin profile is formed as a result of the differing electrophoretic and antigenic properties associated with each of these polymers. Presently, we have examined the capsular polysaccharides of 13 other strains of B. fragilis according to methods used for the prototype strain. The capsules of these strains were extracted, partially purified, and analyzed by immunoelectrophoresis at pH 7.3. Following reaction with homologous polyclonal antisera, each of the capsular preparations tested yielded a complex precipitin profile similar to that of the prototype strain. When reacted by immunoelectrophoresis with polyclonal antiserum to 9343 or with monoclonal antibodies to PS A and PS B, these capsular preparations appeared to be antigenically diverse; some preparations (50%) showed complete or partial cross-reaction. These results suggest that the dual polysaccharide motif seen with the prototype strain is a common feature of B. fragilis strains. In addition, the antigenic heterogeneity of B. fragilis capsular polysaccharides could be used for the development of a serological typing scheme.     

Genetic and phenotypic differences between Legionella pneumophila strains

Legionnaires' disease is a potentially lethal pneumonia that is primarily due to infection by the species Legionella pneumophila, although more than 40 other species are known. Certain L. pneumophila subgroups, particularly serogroup 1, are associated with the majority of the epidemics. The genetic bases for these differences in virulence have not been determined. Three strains, AA100, JR32, and Lp01, have been used in many molecular pathogenesis studies of L. pneumophila. We found genetic differences between these strains by PCR and Southern analyses that may be related to their ability to cause disease. We also examined the distribution of these genetic loci in clinical and environmental isolates of Legionella and found a correlation between the presence of two of these loci, rtxA and lvh, and the ability to cause disease in humans. Examination of the interactions of these strains with host cells suggested that they differ in important phenotypic characteristics including adherence, entry, and intracellular replication. Furthermore, in the mouse model of infection they display differing levels of replication in lungs. These studies emphasize the importance of further investigation into the genetic makeup of these strains, which is likely to lead to the identification of additional factors involved in Legionella pathogenesis.     

Biochemical and immunological differences between hydrophobic and hydrophilic strains of Streptococcus mutans.

Hydrophobic strains of Streptococcus mutans were compared with paired variants showing reduced hydrophobicity. Extracts of hydrophobic cells contained a number of high-molecular-weight proteins which were not present on cells with decreased hydrophobicity. The proteins were found in purified cell walls, suggesting that they are located on the bacterial surface. Trypsin treatment of whole cells destroyed the proteins and reduced the hydrophobicity. Chemical analysis did not reveal any marked differences in the proportion of cell wall constituents. The amino acid compositions and lipoteichoic acid contents of hydrophobic and hydrophilic cell walls were similar. Culture supernatants from the hydrophilic variants contained high-molecular-weight proteins similar to those extracted from the cell walls of the hydrophobic parent strains, indicating that the variants were impaired in their ability to incorporate the hydrophobicity-associated proteins into the cell wall. The dominant protein had a molecular weight of 190,000, similar to that of antigen I/II (B) of S. mutans.     

Selected characteristics of pathogenic and nonpathogenic strains of Bacteroides gingivalis.

Strains of Bacteroides gingivalis were compared for the presence of properties associated with pathogenicity. Some strains were infectious in pure culture in an in vivo model (guinea pig), and all but one of these were more collagenolytic than those which failed to cause lesions in guinea pigs. However, other factors seem to be necessary for the induction of an infection in this animal model.     

Antigenic differences between strains of Newcastle disease virus

Summary Comparison of strains of Newcastle disease virus by kinetic neutralization tests and immunodiffusion tests (after virion disruption by ether and Tween 80) enabled antigenic differences to be demonstrated between four out of five strains tested. No correlation was demonstrated between the antigenic structure and virulence of these strains.With 3 Figures