Optical sensors for detection of bacteria. 1. General concepts and initial development.

The concept of using immobilized nucleic acid stains as detection chemistry to fabricate optical bacterial sensors is first demonstrated. SYTO 13 (a green fluorescent cell stain) is used as the molecular recognition element and fluorescent reporter in the sensor. The sensor responds to aqueous and aerosolized bacterial samples in 15 and 30 min, respectively. In addition, the sensor can discriminate a change in Pseudomonas aeruginosa (Pa) cell concentration of 1 order of magnitude or less and can detect down to 2.4 x 10(5) cells/mL of Pa cells. The utility of the sensor is demonstrated by monitoring the growth of a Pa cell culture over a period of 50 h.

Serum IgG reactivity to subgingival bacteria in initial periodontitis, gingivitis and healthy subjects.

BACKGROUND/AIMS: Established periodontal diseases may be associated with antibody responses to periodontal pathogens, but it is not known at which stage of disease this antibody response is initiated. This study aimed to characterize the host systemic response in initial periodontitis, gingivitis, and periodontal health, to evaluate whether elevated serum antibodies to subgingival species could be detected in initial periodontitis. METHOD: Human systemic immune response were evaluated to 40 subgingival bacterial species in 16 healthy, 21 gingivitis, 11 initial periodontitis and 5 progressing recession adults. Subjects had minimal periodontal attachment level (AL) loss at baseline. Disease categories were determined after 12 months monitoring at three-month intervals. Increased AL loss > or = 1.5 mm (disease activity) at interproximal sites defined initial periodontitis, recession was characterized by AL loss at buccal sites. Serum IgG antibodies were evaluated semi-quantitatively by immunoblot from blood taken at baseline, active and final visits. RESULTS: No antibody was detected from 55% of reactions. When detected, levels were below those reported for advanced periodontitis subjects. There were no major differences in serum antibody levels between healthy, gingivitis and initial periodontitis subjects, despite differences in the subgingival microbiota. Serum antibodies for more species were detected in recession subjects, compared with the other study subjects. No changes in antibody levels were detected between baseline, active, and final visits. No systematic association between species colonization and presence of systemic antibody was observed. CONCLUSIONS: This study did not detect differential elevation of mean serum antibody levels in initial periodontitis subjects, suggesting that serum antibody levels are not sensitive risk markers for initial periodontitis.

Campylobacter species in health, gingivitis, and periodontitis.

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.

Identification of oral Actinomyces species using DNA probes.

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.

Subgingival temperature and microbiota in initial periodontitis.

The association between subgingival temperature, other clinical characteristics, and the subgingival microbiota was examined in adult subjects with initial periodontitis and differing levels of gingival inflammation. 43 subjects were measured at 6 sites per tooth for pocket depth, attachment level, presence of plaque, gingival redness, bleeding on probing and subgingival temperature at 3-month intervals for 1 year. Subgingival plaque was sampled from 15 initial active periodontitis sites (10 subjects), 121 gingivitis, sites (20 subjects) and 202 healthy sites (13 subjects), and included the 5 hottest and 5 coldest sites in each subject. Plaque samples were analyzed for 13 subgingival species using whole-genomic DNA probes. The major influences on the subgingival microbiota were the clinical status of sites, pocket depth, and the presence of supragingival plaque. No significant association between species and site temperature was observed. Initial active sites were associated with Bacteroides forsythus and Campylobacter rectus, and had a higher mean subgingival temperature and deeper mean pocket depth than inactive sites. A weak association between pocket depth and site temperature was noted. The major influence on subgingival temperature of sites was the anterior to posterior anatomical temperature gradient in the mandible and maxilla.

Microbiota of health, gingivitis, and initial periodontitis.

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.

Rapid characterization of periodontal bacterial isolates by using fluorogenic substrate tests.

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.

Helicobacter mustelae isolation from feces of ferrets: evidence to support fecal-oral transmission of a gastric Helicobacter.

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 Helicobacter pylori-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.