Antigenic heterogeneity of Bacteroides intermedius as recognized by monoclonal antibodies.

Four hybrid cell lines secreting monoclonal antibodies against antigens of Bacteroides intermedius were generated by fusing murine NSI cells with splenocytes from a rat immunized with B. intermedius strain OMZ248. An enzyme-linked immunosorbent assay was used to analyze the distribution of the recognized antigens on 39 strains from various Bacteroides species and on 5 strains from other genera. Only Bacteroides species B. intermedius, B. loescheii, B. melaninogenicus, and B. corporis were found to express at least one of the recognized antigens. Strains of the two asaccharolytic black-pigmenting Bacteroides species were negative. Among the strains capable of binding to one or more of the monoclonal antibodies, five groups with different reactivity patterns could be distinguished. Two of the monoclonal antibodies were specific for B. intermedius. The B. intermedius strains were metabolically almost identical, expressed at least three of the recognized antigens, and fell into three distinct antibody reactivity groups, suggesting a tentative separation of this species into three new serogroups. Oral and nonoral isolates of B. intermedius were, however, not distinguished by the monoclonal antibodies. One monoclonal antibody was directed against an antigen strongly expressed on all saccharolytic black-pigmenting Bacteroides strains tested so far, thus confirming the previously noted antigenic relationship between the species which had emerged from the former B. melaninogenicus subsp. intermedius and B. melaninogenicus subsp. melaninogenicus groups.     

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.     

Long-wave UV light fluorescence for identification of black-pigmented Bacteroides spp.

Black-pigmented Bacteroides strains were grown on blood agar, and the colonies were evaluated for fluorescence from long-wave UV light. Most test strains of Bacteroides melaninogenicus subsp. intermedius exhibited a brilliant red fluorescence. B. melaninogenicus subsp. melaninogenicus fluoresced mostly red-orange. Bacteroides asaccharolyticus showed a yellow or red fluorescence. The intensity of the Bacteroides fluorescence weakened when the black pigment of the colonies developed. In contrast, neither young nor old colonies of the oral species Bacteroides gingivalis displayed fluorescence. Since B. gingivalis can produce severe oral infections and also can seed to nonoral sites, awareness of the inability of this organism to fluoresce is important for microbiologists utilizing UV light fluorescence to screen for black-pigmented Bacteroides spp. The present data also indicate that UV light fluorescence may be a rapid method of distinguishing some black-pigmented Bacteroides spp.     

Antigenic characteristics and serological identification of 10 black-pigmented Bacteroides species.

Strains of 10 black-pigmented Bacteroides species were serologically characterized using absorbed and unabsorbed rabbit antisera. An agglutination test using intact cells or heated cells (100 degrees C for 60 min) from each species and unabsorbed antisera revealed only homologous reactions with little or no reactivity in heterologous assays. Immunodiffusion tests using sonicated antigen demonstrated that Bacteroides gingivalis, B. endodontalis, B. asaccharolyticus, B. macacae, and B. levii are antigenically distinct. Strains of B. gingivalis, B. endodontalis, and B. asaccharolyticus were also clearly identified by the indirect immunofluorescent antibody method. B. intermedius, B. corporis, B. loescheii, B. melaninogenicus, and B. denticola possessed common antigens; however, species-specific antigens detectable with immunoabsorbed antisera were also demonstrated. B. intermedius strains isolated from the human oral cavity included at least two serogroups. In each black-pigmented Bacteroides species, lipopolysaccharide constituted one of the species-specific antigens.     

Effect of the presence of black pigmentedBacteroides of differing pathogenicity on the phagocytosis ofEscherichia coli by rat polymorphonuclear leucocytes

An investigation was made of the ability of rat polymorphonuclear leucocytes (PMNLs) to phagocytoseEscherichia coli in the presence of two species of black pigmentedBacteroides (B. melaninogenicus andB. intermedius). When both the bacteria were opsonized together in the presence of normal rat serum,B. melaninogenicus andB. intermedius impaired the phagocytosis ofE. coli significantly. However, the phagocytosis of these black pigmentedBacteroides remained unaffected in the presence ofE. coli. The inhibition of phagocytosis was seen only after the initial first hour of incubation. The inhibition of phagocytosis ofE. coli in the presence ofB. intermedius was more than in the presence ofB. melaninogenicus. The above observation confirms the important role played by black pigmentedBacteroides in experimental mixed infections withE. coli as observed by us earlier.     

Ability of oral bacteria to degrade fibronectin.

The fibronectin-degrading ability of 116, mainly oral, strains was assayed by using plasma-derived fibronectin adsorbed to a polystyrene surface. Ability to degrade fibronectin was revealed in strains of Bacteroides gingivalis, Bacteroides intermedius, Bacteroides loeschii, Staphylococcus aureus, Staphylococcus epidermidis, Peptococcus prevotii, Clostridium sporogenes, and Propionibacterium acnes. The fibronectinolytic activity of subgingival bacteriological samples was found to be related to the presence of B. gingivalis and B. intermedius. In addition, strains of the nonoral Bacteroides species B. asaccharolyticus and B. fragilis showed fibronectin-degrading ability. No such ability was detected in the oral strains tested of Streptococcus, Veillonella, Actinomyces, Lactobacillus, Actinobacillus, Capnocytophaga, Fusobacterium, or Haemophilus species.     

Immunochemical differences between oral and nonoral strains of Bacteroides asaccharolyticus.

We undertook a morphological and immunochemical comparison of purified outer membrane antigens from oral and nonoral isolates of Bacteroides asaccharolyticus. Electron micrographs of thin sections of whole bacteria revealed a compact, electron-dense capsule external to the outer membrane of oral strains. A loose, web-like material was noted on the surface of several nonoral strains that was distinct from the dense capsule seen on oral strains. Polyacrylamide gel electrophoresis showed distinct differences in the protein band pattern between oral and nonoral isolates; sugar composition was similar with a few exceptions. An indirect fluorescent-antibody test utilizing antiserum to a purified capsular antigen from a single oral strain cross-reacted with all of numerous oral and nonoral strains of B. asaccharolyticus, thereby demonstrating a shared antigen that is species specific for B. asaccharolyticus. However, antibodies to an oral strain-derived capsular antigen were detectable by enzyme immunoassay only in serum from rabbits immunized with oral strains. Thus, definite morphological and immunochemical differences were found between oral and nonoral isolates of B.asaccharolyticus.     

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.     

The immunology of fascioliasis: II. Qualitative studies on the precipitin reaction

Antigens prepared from Fasciola spp. precipitated in agar with antisera obtained from mice or rabbits infected with Fasciola hepatica, and with antisera from cattle infected with F. gigantica, or from rabbits immunized with preparations of adult F. hepatica. In the latter there were at least thirty-eight different antibodies, reacting with both protein and species specific non-protein antigens.

The reactions of the antisera from infected animals were less complex. Antisera from infected cattle or mice gave little or no reaction with protein antigens, whereas antisera from infected rabbits reacted predominantly with one of the protein antigens.

Certain non-protein components of the somata or metabolic products of the flukes also reacted with antisera from infected animals. These components were not species specific, and appeared to be haptens as they did not react with antisera from rabbits immunized with killed fluke antigen.

     

Antigens of scrub typhus rickettsiae: separation by polyacrylamide gel electrophoresis and identification by enzyme-linked immunosorbent assay.

Antigens of plaque-purified Rickettsia tsutsugamushi strains Gilliam, Karp, and Kato were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and were analyzed by an enzyme-linked immunosorbent assay. Six antigens were identified in each of the three prototype strains; in strain Gilliam, these antigens were located in the cell envelope fraction of the organisms. Reactivity of these isolated antigens with homologous or heterologous immune sera indicated that different macromolecules existed in all three strains, although they exhibited very similar mobilities during electrophoresis. Antigens of strain Gilliam reacted equally well with antibodies directed against Gilliam, Karp, or Kato rickettsiae. However, strains Karp and Kato each had two distinct antigens which did not react with heterologous antisera. R. tsutsugamushi antigens retained immunogenicity after electrophoresis, and antisera raised against them reacted with intact organisms and exhibited specificity in reactions with isolated antigens.     

API ZYM system for identification of Bacteroides spp., Capnocytophaga spp., and spirochetes of oral origin

A total of 80 oral strains of Bacteroides gingivalis, B. asaccharolyticus, B. melaninogenicus subsp. intermedius, B. melaninogenicus subsp. melaninogenicus, Capnocytophaga, Treponema denticola, and T. vincentii were characterized with the API ZYM system for 19 enzyme activities. Comparison of anaerobic and aerobic incubation with nine reference strains of these organisms showed no important differences. The key differential tests for black-pigmented Bacteroides strains and treponemes of oral origin were trypsin, alpha-glucosidase, and N-acetyl-beta-glucosaminidase. All Capnocytophaga strains produced distinctive aminopeptidase activities but varied in their glycosidic capabilities. The presence of a trypsin-like activity in B. gingivalis, T. denticola, and a group of Capnocytophaga strains may contribute to tissue destruction in periodontal disease.     

Indirect serum haemagglutinating antibody response to black pigmentedBacteroides during experimental pure infections in rats

The humoral immune response during experimental infection with black pigmented bacteroides was studied by the indirect haemagglutination test. BothBacteroides melanimogenicus (ATCC 25845) andB. intermedius (ATCC 25611), grown in semi-solid agar culture, produced pure subcutaneous, intra-abdominal and lung infections. In each of these infection models,B. intermedius was found to be more pathogenic thanB. melaninogenicus on the basis of gross pathology of the lesion, bacteriological and histopathological findings, and the capacity to produce persistent infection. Cytoplasmic extracts of these strains were used as an antigen for the indirect haemagglutination test. In all the infections,B. intermedius provoked a better and higher antibody response than didB. melaninogenicus, suggesting a potent immunogenic property of the former strain. Peak antibody titres in both groups during all the above infections were seen between the 10th and 15th days post infection (p.i.), which was precisely 3–5 days after peak lesion was achieved. A significant IHA antibody titre persisted up to days 30–37 p.i. These findings suggest that the antibodies to the black pigmentedBacteroides are not protective, but may play a role in the pathogenesis of the diseases.     

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.     

Serological identification of oral Bacteroides spp. by enzyme-linked immunosorbent assay

A rapid method for identifying black-pigmented oral Bacteroides spp. is described. Species-specific rabbit antisera to Bacteroides gingivalis, B. intermedius, and B. melaninogenicus were used in an enzyme-linked immunosorbent assay to identify clinical isolates of black-pigmented Bacteroides spp. from humans. The results showed excellent agreement with biochemical identification of B. gingivalis and B. intermedius. Only 36% of the B. melaninogenicus isolates were identified with the enzyme-linked immunosorbent assay, suggesting that this group of black-pigmented Bacteroides spp. is made up of more than one serotype. The serological enzyme-linked immunosorbent assay should enable rapid identification of black-pigmented Bacteroides spp. isolated from sites of oral diseases and may also be used to identify the presence of these organisms in complex bacterial mixtures from oral sites.     

Black-pigmented Bacteroides spp. in the human oral cavity.

Five healthy children under 6 years of age, five healthy adults, and 10 adult periodontitis patients were examined for the prevalence and distribution of black-pigmented Bacteroides in the oral cavity. A total of 13 samples was obtained from each individual, including four supragingival and four subgingival dental plaques, dental occlusal surface, buccal mucosa, dorsal tongue, tonsil, and whole saliva. Black-pigmented Bacteroides were recovered from nine adult periodontitis patients. Healthy adults harbored the organisms in low incidence and proportions, whereas the children exhibited no cultivable black-pigmented Bacteroides. The organisms were isolated in highest proportions from dental plaque, especially subgingival plaque, and from the tonsil area, indicating that these sites constitute the organisms' primary ecological niche in the oral cavity. The predominant isolate was Bacteroides melaninogenicus subsp. intermedius followed by Bacteroides gingivalis and B. melaninogenicus subsp. melaninogenicus. B. melaninogenicus subsp. levii constituted low proportions of supragingival microflora of one adult periodontitis patient. A positive correlation was demonstrated between the proportion of black-pigmented Bacteroides (mainly B. melaninogenicus subsp. intermedius) and both the severity of gingival inflammation and the periodontal pocket depth, suggesting that these organisms may contribute to the pathogenesis of certain forms of periodontal disease.     

Trypanosoma vivax : Soluble Antigens of • and of other Trypanosomes

Precipitins against trypanosomal antigens occurred in serum from Zebu cattle which had been infected for prolonged periods with Trypanosoma vivax transmitted by Glossina morsitans.

Precipitating antisera against Trypanosoma vivax were used to detect complexes of soluble trypanosomal antigens in sera from rats infected with blood-passaged strains of T. vivax, T. gambiense and T. brucei and in sera from goats infected with a cyclically transmitted strain of T. vivax.

The immunological relationships of antigens of these three species of trypanosomes were studied in terms of antisera against T. vivax.

It was found that the antisera contained antibodies which reacted with antigens common to the three species of trypanosomes. The antisera also contained antibodies which reacted with antigens which may be specific to T. vivax.

     

Effects of estradiol and progesterone on Bacteroides melaninogenicus and Bacteroides gingivalis.

Bacteroides melaninogenicus subsp. intermedius increases in the subgingival microflora during pregnancy. These studies evaluated direct interactions between hormonal steroids and oral Bacteroides species. Resting cell suspensions of pure cultures of plaque organisms were incubated anaerobically with [14C]estradiol and [14C]progesterone. Uptake of labeled compound per microgram of bacterial protein was determined by thin-layer chromatography and liquid scintillation counting. B. melaninogenicus subsp. intermedius and B. melaninogenicus subsp. melaninogenicus took up 2.6 x 10(-4) to 5.4 x 10(-4) mumol of estradiol or progesterone per microgram of cell protein. Minimal steroid uptake was observed with B. gingivalis and five other organisms. Uptake of steroids by B. melaninogenicus subsp. intermedius was temperature dependent and resulted in a labeled product as detected on thin-layer chromatography. Growth curves indicated that intermedius and melaninogenicus subspecies of B. melaninogenicus but not B. gingivalis could substitute progesterone or estradiol for vitamin K, an essential growth factor. Growth of B. melaninogenicus subsp. intermedius in steroids was concentration dependent. Addition of fumarate to resting cells of B. melaninogenicus subspecies as well as B. gingivalis increased steroid uptake by 70 to 500% and resulted in the gas-liquid chromatographic detection of succinate. Cultures given fumarate alone or steroids alone produced no succinate. Steroids appeared to directly interact with the fumarate reductase system and foster the growth of B. melaninogenicus subsp. intermedius. This interaction may be of ecological significance.     

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.     

The biochemical properties and antibiotic susceptibility ofBacteroides melaninogenicus

Summary On the basis of pigment production, both strongly fermentative and completely non-saccharolytic anaerobes have been assigned to the same species,Bacteroides melaninogenicus. However, the present study shows thatB. melaninogenicus is a homogeneous group of strictly anaerobic gram-negative non-sporing rods which produce a black pigment on certain media, but can be identified and differentiated by their biochemical properties without taking pigment production into account. According to the results obtained with ten strains,B. melaninogenicus is characterized by the production of acetic, propionic, isobutyric, butyric, and isovaleric acids in peptone-yeast extract-glucose media, the inability to produce ammonia or propionate from threonine, and the absence of glutamate decarboxylase activity.B. melaninogenicus produces indole, liquefies gelatin, and is non-saccharolytic, as indicated by pH values of 6.2 to 6.6 in media containing 1% of glucose or other carbohydrates. According to the results obtained in tube dilution tests with six strains,B. melaninogenicus is sensitive to penicillins, cephalosporins, bacitracin, chlortetracyclin, chloramphenicol, erythromycin, and rifampicin and resistant to streptomycin, colistin, polymyxin B, and neomycin.

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Zusammenfassung Auf Grand der Pigmentbildung sind sowohl kohlenhydratfermentierende wie auch asaccharolytische Anaerobier als Angehörige der SpeciesBacteroides melaninogenicus eingeordnet worden. Die vorliegenden Untersuchungen an 10 Stämmen zeigen jedoch, daßB. melaninogenicus aus einer homogenen Gruppe strikt anaerober gramnegativer sporenloser Stäbchen besteht, die ohne Berücksichtigung der Pigmentbildung lediglich auf Grund ihrer biochemischen Eigenschaften identifiziert und differenziert werden können.B. melaninogenicus ist durch die Bildung von Essig-, Propion-, Isobutter, Butter- und Isovaleriansäure in Pepton-Hefeextrakt-Glucose-Medien sowie durch Fehlen der Threoninspaltung und der Glutaminatdecarboxylase-Aktivität gekennzeichnet. Die Species bildet Indol und verflüssigt Gelatine. In 1% Glucose oder andere Kohlenhydrate enthaltenden Medien werden pH-Werte von 6,2–6,6 beobachtet, so daß die Species als asaccharolytisch gelten muß. In Reihenverdünnungsversuchen mit 6 Stämmen erwies sichB. melaninogenicus als empfindlich gegen Penicilline, Cephalosporine, Bacitracin, Chlortetracyclin, Chloramphenicol, Erythromycin und Rifampicin sowie als resistent gegen Streptomycin, Colistin, Polymyxin B und Neomycin.

     

Degradation of immunoglobulins A2, A2, and G by suspected principal periodontal pathogens.

Attention has recently been focused on immunoglobulin A1 (IgA1) protease production as a possible virulence factor of bacteria implicated in meningitis and gonorrhea. This report demonstrates that suspected principal etiological agents in destructive periodontal disease include bacteria capable of degrading IgA1, IgA2, and IgG. Representative strains of Bacteroides melaninogenicus subsp. melaninogenicus and Capnocytophaga cleaved IgA1 but not IgA2 in the hinge region to yield intact Fab and Fc fragments. All Capnocytophaga strains also cleaved IgG in the same way. The majority of strains of Bacteroides asaccharolyticus and B. melaninogenicus subsp. intermedius caused complete degradation of both IgA1 and polyclonal IgG. However, some strains left the Fc part of IgA1 intact. Several strains were also capable of completely decomposing IgA2 and S-IgA. Significant IgA-cleaving enzyme activity was detected in whole subgingival dental plaque collected from patients with destructive periodontal disease. The results indicate that colonization of the subgingival area by B. asaccharolyticus, B. melaninogenicus, and Capnocytophaga spp. can induce a local paralysis of the immune defence mechanisms, thereby facilitating the penetration and spread of potentially toxic substances, lytic enzymes, and antigens released by the entire subgingival microflora.