The arbitrary primer polymerase chain reaction (AP-PCR) and Southern blot restriction fragment length polymorphism (RFLP) were used to genotype the periodontal pathogen A. actinomycetemcomitans. Total genomic DNA from 73 strains was extracted by conventional methods. Three random-sequence 10-base oligonucleotide primers were chosen for AP-PCR. The amplified DNA products were separated electrophoretically in a 1% agarose gel containing ethidium bromide and the banding patterns were compared among different strains. For RFLP analysis, DNA was digested with EcoRI, separated on a 0.8% agarose gel and transferred to a nylon membrane. The membrane was probed with a previously characterized 5.2 kilobases (kb) DNA fragment cloned from A. actinomycetemcomitans strain Y4. The probe was labeled with digoxigenin, and hybridized fragments were detected with anti-digoxigenin antibody. AP-PCR produced 4–10 DNA bands in the 0.5–5 kb regions and distinguished 9, 13 or 17 genotypes, depending on the specific primer used. Southern blot RFLP analysis revealed 12 hybridization patterns consisting of 1 or 2 DNA fragments (2–23 kb). The addition of the Southern blot analysis to the AP-PCR analysis gave rise to a total of 30 DNA profiles among the 73 A. actinomycetemcomitans study strains. The results indicate that both AP-PCR and Southern blot analysis are useful in clonal analysis of A. actinomycetemcomitans.
Actinobacillus actinomycetemcomitans; DNA fingerprinting; polymerase chain reaction; restriction fragment length polymorphism; DNA probe; Southern blot
Actinobacillus acrinomycetemcomirans isolates from periodontal pockets were examined for restriction fragment-length polymorphism using a characterized 4.7-kb DNA probe. A total of 6 patterns of RFLP was found in 133 isolates originating from 12 subjects. No relatedness was found between RFLP types and serotypes. Different periodontal sites within the same subject and different individuals within the same family sometimes showed only one type of A. actinomycetemcomitans RFLP. When members among the same family showed 2 RFLP types, children were always infected with the A. acfinomycefemcomitans strains found in at least one of the parents. These findings support the concept of familial spread of A. actinomycetemcomitans. A. actinomycetemcomitans RFLP type B, corresponding to reference strain JP2, seems to be particularly virulent, as indicated from the presence of RFLP type B in 3 subjects who converted from a healthy periodontal state to localized juvenile periodontitis. RFLP type B was not detected in any of the 21 A. acrinomycetemcomitans-infected patients with adult periodontitis. The RFLP method seems to be useful in determining the epidemiology and possibly the potential virulence of periodontal strains of A. actinomycetemcomitans.
juvenile periodontitis; epidemiology; virulence; restriction fragment-length polymorphism; DNA probes; Actinobaccillus actinomycetemcomitans
Dialister pneumosintes is a nonfermentative, anaerobic, gram-negative rod that grows with small, circular, transparent, shiny, smooth colonies on blood agar. Even though D. pneumosintes has been recovered from deep periodontal pockets, little is known about the relationship between the organism and destructive periodontal disease. This study describes a rapid PCR method to identify D. pneumosintes in periodontal samples. The PCR identification method detected as little as 10 pg of D. pneumosintes DNA or about 1 to 10 cells without nonspecific amplification of various periodontopathic bacteria. Twelve of 22 subgingival samples from adult periodontitis lesions yielded D. pneumosintes either by culture or by PCR identification. In culture-positive samples, D. pneumosintes averaged 3.9% (0.001 to 10.8%) of total isolates. Studies are needed to delineate virulence factors of D. pneumosintes pertinent to periodontal disease.
A geographically homogeneous population of 83 subjects, from 21 families with localized juvenile periodontitis (LJP), and 35 healthy control subjects was monitored, over a 5-year period, for the presence of the periodontal pathogen Actinobacillus actinomycetemcomitans. Restriction fragment length polymorphism (RFLP) analysis was used to monitor the distribution of genetic variants of this bacterium in LJP-susceptible subjects that converted from a healthy to a diseased periodontal status. A. actinomycetemcomitans was cultured from 57% of the LJP family members accessioned into the study. Nine of 36 LJP-susceptible subjects, in seven families, developed signs of periodontal destruction. All but one of these conversion subjects harbored A. actinomycetemcomitans. Bacterial variants representative of a single RFLP group (II) showed the strongest correlation with conversion (P < 0.002). Six of nine conversion subjects were infected with A. actinomycetemcomitans from this group. RFLP group II variants also prevailed in 8 of 22 probands but were absent in the 35 healthy control subjects. In contrast to the selective distribution of group II variants is diseased individuals, variants belonging to RFLP groups XIII and XIV were found exclusively in the control subjects. Thus, the use of RFLP to type clinical isolates of A. actinomycetemcomitans has resulted in the identification of genetic variants that predominate in LJP and health. These results indicate that studies concerned with the pathogenicity of this bacterium in LJP should be focused on the group II variants.
In this study, we have assessed four strains of Prevotella intermedia, isolated from periodontally involved lesions, for their ability to inhibit lymphocyte functions. All four strains were found to cause a dose-dependent inhibition of B- and T-cell proliferation in response to mitogens and antigens. This was reflected in altered DNA, RNA, and protein syntheses. Furthermore, P. intermedia appeared to affect the early stages of cell activation. This was ascertained by kinetic analysis in which it was determined that the extract had to be present during the first 24 h of incubation to cause suppression. Moreover, direct assessment of the early stages of cell activation indicated that release of cytokines and expression of the interleukin 2 receptor and CD69 on T cells were inhibited by P. intermedia sonic extracts. Finally, preliminary characterization of the immunosuppressive agent indicates that it has a molecular mass of approximately 50 kDa and is heat labile. It has been proposed that impaired host defense may play a pivotal role in the pathogenesis of many infections. The data presented in this paper suggest that microbially mediated immunosuppression may contribute to the pathogenesis of periodontal disease by altering the nature and consequences of host-parasite interactions.
The hemagglutinating factor (hemagglutinin) of Bacteroides gingivalis was prepared from the supernatant of a 5-day diffusate broth culture by ammonium sulfate precipitation and column chromatography with a hydrophobic column of Phenyl-Sepharose CL-4B, DEAE-Sephadex A-50, and Sephadex G-100 gel filtration. The hemagglutinating activity of the preparation was 53.3 times higher than that of ammonium sulfate precipitate. In electron microphotographs, hemagglutinin appears to have a vesicle or tubelike structure. The hemagglutinating activity of intact cells was completely destroyed by heating at 100 degrees C for 10 min, but the activity of extracted hemagglutinin was heat stable. The activity of hemagglutinin was inhibited by L-arginine and L-lysine and partially inhibited by phospholipase D, but it was not affected by proteolytic enzymes, neuraminidase, hyaluronidase, lipase, phospholipase A and C, or sugars. The B. gingivalis hemagglutinin appeared to be comprised mainly of a 40,000-molecular-weight material. The Fab fragment of immunoglobulin G prepared from rabbit antiserum to whole cells of B. gingivalis and monoclonal antibody against the hemagglutinin bound to the cell surface and inhibited the hemagglutinating activity of both the cells and the purified hemagglutinin.
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.
We developed a medium for the selective recovery of Haemophilus aphrophilus. The medium, designated TSBVF, was composed of 4% tryptic soy agar, 10% heat-inactivated horse serum, 75 micrograms of bacitracin per ml, 5 micrograms of vancomycin per ml, and 50 micrograms of sodium fluoride per ml. TSBVF yielded a threefold higher recovery of oral H. aphrophilus than did chocolate agar with 75 micrograms of bacitracin per ml, which is a medium routinely used to diagnose human Haemophilus infections. H. aphrophilus and the few contaminating organisms on TSBVF were readily distinguished on the basis of colony morphology. The H. aphrophilus isolates exhibited variable fermentation of raffinose and dextrin but otherwise were biochemically similar. In a clinical study, H. aphrophilus was frequently recovered from supragingival plaque and saliva and occasionally from buccal mucosa and the tonsils. It was also isolated from 29 of 56 subgingival sites in 11 of 14 subjects. Its proportion of the subgingival microflora averaged 0.13% for healthy periodontal sites, 0.05% for adult periodontitis lesions, and 0.03% for localized juvenile periodontitis lesions. We concluded that H. aphrophilus is an indigenous bacterium of the human oral cavity. It occurs in low proportions in subgingival plaque and plays no apparent role in advanced periodontal disease in humans.
An immunofluorescence technique was developed for the in situ identification of specific bacteria in marine microfouling films. Microorganisms adherent to glass plates after 30 days of immersion in a synthetic seawater system were cultured and classified by biochemical tests, flagellar arrangement, and the API 20E system. All isolates were gram-negative aerobic or facultative motile rods, predominantly Pseudomonas spp. Rabbit antisera to the five dominant organisms including Achromobacter spp., Comamonas terrigena, P. putrefaciens, a yellow-pigmented Pseudomonas sp., and Vibrio alginolyticus were prepared. These antisera were shown to be species specific in indirect immunofluorescence assays against a battery of 26 marine isolates from 14 bacterial species, with the exception of antisera to the Pseudomonas spp, which cross-reacted with each other but not with test bacteria of other genera. These immunofluorescent reagents enabled the in situ identification of all five bacterial species in microfouling films. Low-surface-energy test plates had smaller numbers of adherent bacteria in microfouling films than medium-surface-energy test plates, suggesting that the degree of microfouling may be influenced by the surface energy. In addition, the reagents could identify up to 39% of the attached bacteria in microfouling films spontaneously formed on steel plates in flow cells deployed in different areas of the Atlantic Ocean. The microbial composition of the ocean-formed films varied with the geographical area of their formation. The present results indicate that immunofluorescence techniques may provide a rapid and reliable means to identify, in situ, specific bacteria in marine microfouling films.
The serotype c antigen from Actinobacillus actinomycetemcomitans was purified with fractional ethanol precipitation of cell-free culture supernatant, sequential ion-exchange chromatography, and gel filtration chromatography. The preparation obtained demonstrated a single precipitin line in immunodiffusion, immunoelectrophoresis, and crossed immunoelectrophoresis when rabbit antisera to serotype c whole bacterial cells were used. No immunological reaction was detected with antisera to serotype c lipopolysaccharide, indicating that lipopolysaccharide was not present in the preparation. The serotype c antigen was composed of 95% carbohydrate, 2% protein, and 3.1% phosphate. Gas chromatographic analysis of the antigen obtained from growth in either complex or chemically defined media revealed that the carbohydrate constituent was composed of 84 to 90.1% mannose, 4.8 to 16% glucose, 1.9% N-acetylglucosamine, 1.4% fucose, and 0.2% galactose. The present data suggest that A. actinomycetemcomitans serotype c antigen is predominantly a mannose-containing carbohydrate suggestive of a mannan.
The minimal inhibitory concentrations of a series of antimicrobial agents for human oral organisms were determined under anaerobic growth conditions by an agar dilution assay. With the exception of black-pigmented Bacteroides spp., minimal inhibitory concentrations for oral isolates were similar to those for non-oral isolates of organisms of the same or closely related species.
Actinobacillus actinomycetemcomitans from the human oral cavity was serologically characterized with rabbit antisera to the type strain NCTC 9710; a number of reference strains, including Y4, ATCC 29522, ATCC 29523, ATCC 29524, NCTC 9709; and our own isolates representative of each of 10 biotypes. Using immunoabsorbed antisera, we identified three distinct serotypes by immunodiffusion and indirect immunofluorescence. Serotype a was represented by ATCC 29523 and SUNYaB 75; serotype b was represented by ATCC 29522 and Y4; and serotype c was represented by NCTC 9710 and SUNYaB 67. Indirect immunofluorescence revealed no reaction between the three A. actinomycetemcomitans serotype-specific antisera and 62 strains representing 23 major oral bacterial species. Distinct from the serotype antigens were at least one A. actinomycetemcomitans species common antigen and an antigen shared with other Actinobacillus species, Haemophilus aphrophilus, and Haemophilus paraphrophilus. All serotype a A. actinomycetemcomitans strains failed to ferment xylose, whereas all serotype b organisms fermented xylose. Serotype c included xylose-positive as well as xylose-negative strains. A total of 301 isolates of A. actinomycetemcomitans from the oral cavity of 74 subjects were serologically categorized by indirect immunofluorescence with serotype-specific rabbit antisera. Each patient harbored only one serotype of A. actinomycetemcomitans. Fourteen healthy subjects, five diabetics, and seventeen adult periodontitis patients exhibited serotypes a and b in approximately equal frequency, whereas serotype c was found less frequently. In contrast, in 29 localized juvenile periodontitis patients, the incidence of serotype b was approximately two times higher than that of serotypes a or c, suggesting a particularly high periodontopathic potential of A. actinomycetemcomitans serotype b strains. In subjects infected with A. actinomycetemcomitans, serum antibodies were detected to the serotype antigens, indicating that these antigens may play a role in the pathogenesis of periodontal disease.
The promyelocytic HL-60 cell line was examined for susceptibility to leukotoxin from Actinobacillus actinomycetemcomitans. Strains of A. actinomycetemcomitans which caused lysis of human peripheral blood polymorphonuclear leukocytes also lysed HL-60 cells as determined by release of intracellular lactate dehydrogenase. The killing of HL-60 cells by A. actinomycetemcomitans was dose dependent and temperature dependent, reached maximal levels after 45 min of incubation, and was inhibited by rabbit antisera to A. actinomycetemcomitans. Of 100 oral isolates of A. actinomycetemcomitans from 55 subjects, 16% from 11 healthy subjects, 43% from 13 adult periodontitis patients, 75% from 4 insulin-dependent diabetics, 66% from 2 generalized juvenile periodontitis patients, and 55% from 25 localized juvenile periodontitis patients produced leukotoxin. The same subject could harbor both leukotoxin-producing and -nonproducing isolates. The significantly higher proportion of leukotoxin-producing isolates in the disease groups compared with the healthy group is consistent with the hypothesis that leukotoxin from A. actinomycetemcomitans is an important virulence factor in the pathogenesis of certain forms of periodontal disease.
On the basis of in vitro susceptibility testing of antibiotics, dyes, and other antimicrobial agents, we developed and evaluated a medium, TBBP, for the selective isolation of oral Capnocytophaga spp. TBBP medium consists of 4% Trypticase soy agar (BBL Microbiology Systems, Cockeysville, Md.), 5% sheep blood, 0.1% yeast extract, 50 micrograms of bacitracin per ml, and 100 micrograms of polymyxin B per ml. A total of 34 Capnocytophaga stock cultures grew well on TBBP medium. Except for some streptococcal strains, TBBP medium inhibited growth of all test stock culture isolates of common oral gram-positive and gram-negative species. In a clinical study of 15 deep periodontal pockets, TBBP medium demonstrated Capnocytophaga recoverability that was similar to or higher than that shown by a nonselective blood agar medium. Typical Capnocytophaga colonial morphology enabled us to readily distinguish this organism from the few other bacteria which could grow on TBBP medium.
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.
A selective medium, TSBV (tryptic soy-serum-bacitracin-vancomycin) agar, was developed for the isolation of Actinobacillus actinomycetemcomitans, TSBV agar contained (per liter) 40 g of tryptic soy agar, 1 g of yeast extract, 100 ml of horse serum. 75 mg of bacitracin, and 5 mg of vancomycin. The TSBV medium suppressed most oral species and permitted significantly higher recovery of A. actinomycetemcomitans than nonselective blood agar medium. The distinct colonial morphology and positive catalase reaction of A. actinomycetemcomitans easily distinguished this bacterium from Haemophilus aphrophilus, Capnocytophaga species, and a few other contaminating organisms. With the TSBV medium, even modestly equipped laboratories will be able to isolate and identify A. actinomycetemcomitans from clinical specimens.
The cellular fatty acid and protein content of twenty-five representative strains of Actinobacillus actinomycetecomitans isolated from juvenile and adult periodontitis patients was compared to that of 15 reference strains of oral and nonoral Actinobacillus species and Haemophilus aphrophilus. Trimethylsilyl derivatives of the fatty acid methyl esters were analyzed by gas-liquid chromatography. The predominant fatty acids of all 40 strains examined were 14:0, 3-OH 14:0, 16 delta, and 16:0. Actinobacillus seminis (ATCC 15768) was unlike the other strains examined because of a greater amount of 14:0 detected. The soluble protein analysis using polyacrylamide gel electrophoresis revealed that A. actinomycetemcomitans, H. aphrophilus, and nonoral Actinobacillus species possessed distinct protein profiles attesting to the validity of separating these organisms into different species. Established biotypes of A. actinomycetemcomitans could not be differentiated on the basis of fatty acid or protein profiles.
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.
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.
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.
The agar dilution technique was used for determination of the antibiotic susceptibilities of 57 oral isolates and 2 nonoral isolates of Actinobacillus actinomycetemcomitans. Tetracycline, minocycline, and chloramphenicol inhibited more than 96% of the strains tested at a concentration of less than or equal to 2 micrograms/ml; 89% of the strains were inhibited by 2 micrograms of carbenicillin per ml. The other antimicrobial agents tested were less active. Approximately 10% of the A. actinomycetemcomitans strains were resistant to ampicillin, erythromycin, and penicillin G at concentrations of 32 to 64 micrograms/ml. These data suggest that tetracycline and minocycline may be valuable drugs in the treatment of A. actinomycetemcomitans infections.
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.
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.
Macaca arctoides monkeys develop periodontal disease, and they harbor a periodontopathic indigenous flora largely similar to that of humans. This study showed that various Haemophilus isolates and H2O2-splitting asaccharolytic Bacteroides melaninogenicus strains constituted major segments of the monkey periodontal microflora. These organisms have not been previously identified among human isolates. Furthermore, the present data revealed that asaccharolytic B. melaninogenicus strains increased in proportion from a few percent to about 66% of the total isolates concomitant with the development of a significant loss of alveolar bone mass. Hence, this study strongly implicates B. melaninogenicus subsp. asaccharolyticus and closely related strains as important pathogens in actively destructive periodontal disease.