This study compared the subgingival microbiota in periodontal health, gingivitis and initial periodontitis using predominant culture and a DNA probe, checkerboard hybridization method. 56 healthy adult subjects with minimal periodontal attachment loss were clinically monitored at 3-month intervals for 12 months. More sites demonstrated small increments of attachment loss than attachment gain over the monitoring period. Sites, from 17 subjects, showing > or = 1.5 mm periodontal attachment loss during monitoring were sampled as active lesions for microbial analysis. Twelve subjects demonstrated interproximal lesions, and 5 subjects had attachment loss at buccal sites (recession). Cultural studies identified Bacteroides forsythus, Campylobacter rectus, and Selenomonas noxia as the predominant species associated with active interproximal lesions (9 subjects), whereas Actinomyces naeslundii, and Streptococcus oralis, were the dominant species colonizing buccal active sites. A. naeslundii, Campylobacter gracilis, and B. forsythus (at lower levels than active sites) were the dominant species cultured from gingivitis (10 subjects). Health-associated species (10 subjects) included Streptococcus oralis, A. naeslundii, and Actinomyces gerencseriae. DNA probe data identified higher mean levels of B. forsythus and C. rectus with active (7 subjects) compared to inactive periodontitis sites. Porphyromonas gingivalis and Actinobacillus actinomycetemcomitans were detected infrequently. Cluster analysis of the cultural microbiota grouped 8/9 active interproximal lesions in one subcluster characterized by a mostly gram-negative microbiota, including B. forsythus and C. rectus. The data suggest that B. forsythus C. rectus and S. noxia were major species characterizing sites converting from periodontal health to disease. The differences in location and microbiota of interproximal and buccal active sites suggested that different mechanisms may be involved in increased attachment loss.

At least seven Campylobacter species have been identified from subgingival sites. Campylobacter rectus has been implicated as a periodontal pathogen; however, association with periodontal infections of other Campylobacter species, especially the newly described Campylobacter showae, is unclear. This study examined which Campylobacter species were associated with periodontal health and disease. Subgingival Campylobacter species from initial and established periodontitis were compared with species from periodontally healthy subjects, including subjects with gingivitis. Campylobacter species were isolated on selective media and identified by whole-cell protein profiles (SDS-PAGE). Except for C. rectus, Campylobacter levels were frequently below the detection limit (2-5% of the microbiota) of non-selective culture methods. C. rectus and C. showae, including Campylobacter X, were found more frequently and in higher levels from diseased than from healthy periodontal sites. C. gracilis was the dominant Campylobacter species found in relatively shallow pockets; however, its presence was unrelated to periodontal health or disease. C. concisus was isolated in higher proportions from relatively shallow and healthy sites, compared with deeper pockets. C. curvus was unrelated to periodontal health or disease. Analysis of the study data confirmed the relationship of C. rectus with diseased subgingival sites and indicated that C. showae may also be associated with periodontal disease.

Helicobacter mustelae has been isolated from stomachs of ferrets with chronic gastritis and ulcers. When H. mustelae is inoculated orally into H. mustelae-negative ferrets, the animals become colonized and develop gastritis, a significant immune response, and a transient hypochlorhydria. All of these features mimic Helicobacterpylori-induced gastric disease in humans. Because the epidemiology of H. pylori infection is poorly understood and its route of transmission is unknown, the feces of weanling and adult ferrets were cultured for the presence of H. mustelae. H. mustelae was isolated from the feces of 11 of 36 ferrets by using standard helicobacter isolation techniques. H. mustelae was identified by biochemical tests, ultrastructural morphology, reactivity with specific DNA probes, and 16S rRNA sequencing. H. mustelae was not recovered from 20-week-old ferrets which had been H. mustelae positive as weanlings, nor was H. mustelae recovered from 1-year-old ferrets. Isolation of H. mustelae from feces may correspond to periods of transient hypochlorhydria, or H. mustelae may be shed in feces intermittently. The H. mustelae-colonized ferret provides an ideal model for studying the pathogenesis and transmission of H. pylori-induced gastric disease.

Eighty-nine species of subgingival bacteria, represented by 121 reference strains and 892 patient isolates, including gram-negative, gram-positive, aerobic, facultatively anaerobic, microaerophilic, and anaerobic species, were characterized with a panel of fluorogenic, 4-methylumbelliferyl-linked substrate tests. Identifications of all patient isolates were confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of whole-cell proteins relative to reference strains. Characteristic profiles of positive fluorogenic reactions differentiated most of the species, including five Porphyromonas species, six pigmenting and five nonpigmenting Prevotella species, Bacteroides forsythus, three Capnocytophaga species, six Actinomyces species, four Propionibacterium species, and eight Streptococcus species. Two mannoside isomers differentiated Actinomyces israelii and Actinomyces gerencseriae. In addition to Porphyromonas gingivalis, B. forsythus, and Capnocytophaga species, Fusobacterium alocis, Actinomyces odontolyticus, Actinomyces meyeri, and Bifidobacterium dentium were all positive for so-called trypsin-like activity. Fusobacterium nucleatum, Eikenella corrodens, Actinobacillus actinomycetemcomitans, and Campylobacter species were nonreactive with the carbohydrate-based substrates tested. Fluorogenic substrate tests provided a sensitive and simple method for biochemical characterization that could presumptively identify to species level most subgingival isolates within 4 h. The method was ideal for rapidly obtaining presumptive identifications of isolates prior to confirming identifications by definitive methods, such as SDS-PAGE.

Oral Actinomyces comprise a major segment of both the supra- and subgingival microbiota; however, little is known about the distribution of individual species in different sites or clinical conditions. The purpose of the present investigation was to develop DNA probes for suggested species and genotypes of oral Actinomyces. Whole genomic DNA probes to 12 human oral species and/or serotypes were labeled with digoxigenin and used to seek cross-reactions among the taxa using the checkerboard DNA-DNA hybridization assay. The Actinomyces formed three distinct groups: 1) Actinomyces georgiae, Actinomyces meyeri and Actinomyces odontolyticus serotypes I and II; 2) Actinomyces viscosus and Actinomyces naeslundii serotypes I, II, III and WVA 963; and 3) Actinomyces gerencseriae and Actinomyces israelii. Cross-reactions among taxa were detected and minimized by increasing the temperature of the post-hybridization high-stringency wash to 80 degrees C. Despite the elevation in high stringency wash temperature, cross-reactions among strains of the A. naeslundii/A. viscosus group persisted. Probes for two of the three currently recognized genospecies in this group were prepared by removing the DNA in common between cross-reacting species using subtraction hybridization and polymerase chain reaction. Nine species and genospecies could be clearly separated by a combination of whole genomic and subtraction hybridization probes and by increasing the high-stringency wash temperature. A total of 195 fresh isolates of Actinomyces were grouped in a blind study using DNA probes and separately by SDS-PAGE protein profiles. Concordance between the two methods was 97.3%. The probes and hybridization conditions were tested for their ability to detect the Actinomyces species and genospecies in samples of supragingival and subgingival plaque from periodontitis subjects using checkerboard DNA-DNA hybridization. The probes detected the species in samples of supragingival and subgingival plaque. We concluded that whole genomic and subtraction hybridization DNA probes facilitate the detection and enumeration of species and genospecies of Actinomyces in plaque samples.