Amino acid utilization by Fusobacterium nucleatum grown in a chemically defined medium.

The present investigation evaluated amino acid utilization by 120 strains of Fusobacterium nucleatum in a chemically defined medium and attempted to relate the patterns to 3 proposed subspecies of F. nucleatum. Strains were inoculated into a chemically defined medium, with and without 2 g/l glucose, consisting of 14 inorganic salts, 21 amino acids, 23 vitamins and cofactors, and 7 purines and pyrimidines. After 7 days of anaerobic incubation, the spent culture medium, as well as the uninoculated control medium, were analyzed for amino acid content by ion chromatography. Amino acid utilization was determined by the differences in concentrations of amino acids found in inoculated and uninoculated samples. If greater than 34% of the amino acid was removed from the medium, the amino acid was considered to be utilized. Of the 21 amino acids present in the chemically defined medium, 8 amino acids, lysine, glutamine, asparagine, histidine, threonine, serine, glutamate and cysteine were consistently utilized. Four amino acids, tyrosine, tryptophan, methionine and aspartate were utilized by some strains but not others. Nine amino acids, alanine, leucine, isoleucine, glycine, valine, phenylalanine, proline, ornithine, and arginine were not utilized by any of the strains. The utilization patterns did not relate to subspecies formed on the basis of SDS-PAGE and DNA hybridization.

Proposal of three subspecies of Fusobacterium nucleatum Knorr 1922: Fusobacterium nucleatum subsp. nucleatum subsp. nov., comb. nov.; Fusobacterium nucleatum subsp. polymorphum subsp. nov., nom. rev., comb. nov.; and Fusobacterium nucleatum subsp. vincentii subsp. nov., nom. rev., comb. nov.

Heterogeneity among isolates of Fusobacterium nucleatum has been recognized for many years. The phenotypic properties of 340 strains considered to be F. nucleatum were examined. While these strains were phenotypically similar and fit the description of F. nucleatum, they could be differentiated into three groups on the basis of electrophoretic patterns of whole-cell proteins and DNA homology. Strains in groups I and II showed greater than 80% DNA homology within groups and less than 75% similarity between groups. Strains of group III demonstrated greater than 85% DNA homology to each other and less than 65% similarity to strains in groups I and II. We propose that Fusobacterium nucleatum be divided into the following three subspecies: Fusobacterium nucleatum subsp. nucleatum, with type strain ATCC 25586; Fusobacterium nucleatum subsp. polymorphum, with type strain ATCC 10953; and Fusobacterium nucleatum subsp. vincentii, with type strain ATCC 49256.

The predominant cultivable microbiota of crevicular epithelial cells.

The purpose of the present investigation was to compare the composition of the predominant cultivable microbiota associated with gingival crevicular epithelial cells with that of the unattached microbiota recovered from the same site. Samples were taken from 2 diseased sites from 8 periodontal patients, by scraping the epithelial lining of the pocket with a curette. The epithelial cells were separated from the unattached subgingival bacteria by centrifugation in a reduced 50% Percoll density gradient. Epithelial cells formed a band at the top of the gradient and were removed separately from the unattached bacteria located at the base. Each layer was dispersed, diluted and plated on Trypticase soy agar with 5% sheep blood and 50 isolates were characterized from each sample. The microorganisms associated with the epithelial layer harbored 5- to 20-fold higher mean percentages of Bacteroides gingivalis, Bacteroides intermedius and Peptostreptococcus micros. The layer of unattached organisms exhibited 4- to 10-fold higher mean percentages of Streptococcus uberis, Capnocytophaga ochracea, Eikenella corrodens and Veillonella parvula.

Relationship of subgingival microbial complexes to clinical features at the sampled sites.

Subgingival plaque samples from 100 active destructive periodontal lesions and 150 inactive subgingival sites in 33 subjects were analyzed by predominant cultivable microbiota techniques. 50 isolates were characterized from each sample and where possible, the isolate was placed in 1 of 134 microbial species or groups. The sites were clustered on the basis of the proportions of all of the species detected in each sample using a minimum similarity matching coefficient and an average unweighted linkage sort. 10 clusters containing 166 sites were formed which exhibited greater than 35% minimum similarities. All clusters were made up of sites from multiple subjects and were formed on the basis of different combinations of micro-organisms. Certain complexes of micro-organisms appeared to relate to the severity of periodontal destruction and the activity of the sampled site. The combination of F. nucleatum, B. forsyth and W. recta (cluster VII) or B. gingivalis, B. intermedius and S. intermedius (cluster VIII) distinguished clusters made up of sites which on average had the most attachment loss and the deepest pockets. These clusters contained the highest proportions of active sites and sites which lost greater than 3 mm of attachment after therapy. Clusters dominated by V. parvula (cluster III), the Actinomyces sp. (cluster X) or the combination of S. sanguis II, S. mitis, V. parvula and S. intermedius (cluster II) were made up of sites which exhibited less active disease and responded more favorably to therapy. Sites in other clusters exhibited moderate levels of prior destructive disease and disease activity status closer to the mean values for all 250 sites.(ABSTRACT TRUNCATED AT 250 WORDS)

Clinical, microbiological and immunological features of subjects with refractory periodontal diseases.

27 subjects with active destructive periodontal diseases were treated by modified Widman flap surgery and systemic tetracycline and divided into 4 groups based on pre- and post-therapy hazard rates (% of sites losing greater than 3 mm of attachment in 1 year). Pre- and post-therapy hazard rates were respectively: group I (3 subjects) less than 4 and less than 4; group II (8 subjects) greater than 4 and less than 4; group III (3 subjects) less than 4 and greater than 4; group IV (refractory group of 13 subjects) greater than 4 and greater than 4. Baseline mean pocket depths and attachment loss of groups I and II subjects were less than groups III and IV subjects and exhibited less suppuration. 6 group IV subjects lost a total of 38 teeth after therapy, in contrast to no tooth loss in subjects in the other 3 groups. Redness, bleeding on probing, plaque levels and age did not differ among groups. Subjects in the 4 groups differed in the subgingival species to which they showed elevated serum antibody responses. Group IV subjects showed elevated responses to a select range of gram-negative species, including A. actinomycetemcomitans strains Y4 or ATCC 29523, F. nucleatum and B. intermedius. No subject in any of the other groups exhibited an elevated response to B. intermedius. The mean % of each species in all sampled sites, both before and after therapy, was computed for each subject. Subjects in groups III and IV (high post-therapy hazard rates) exhibited elevated mean levels of B. forsythus, F. nucleatum, S. intermedius, E. corrodens, and B. gingivalis.(ABSTRACT TRUNCATED AT 250 WORDS)

The predominant cultivable microbiota of active and inactive lesions of destructive periodontal diseases.

Subgingival plaque samples were taken from active and inactive lesions in 33 subjects exhibiting active destructive periodontal diseases. Active diseased sites were those which showed a significant loss of attachment within a 2-month interval as computed by the "tolerance method". The predominant cultivable species from 100 active sites were compared with those found in 150 inactive sites of comparable pocket depth and attachment level loss. Among the 33 subjects, W. recta, B. intermedius, F. nucleatum, B. gingivalis and B. forsythus were elevated more often in active sites; whereas, S. mitis, C. ochracea, S. sanguis II, V. parvula and an unnamed Actinomyces sp. were elevated in inactive sites. The likelihood of a site being active was increased if B. forsythus, B. gingivalis, P. micros, A. actinomycetemcomitans, W. recta, or B. intermedius were detected in that site, and decreased if S. sanguis II, the Actinomyces sp., or C. ochracea were detected.

Clinical, microbiological and immunological features of subjects with destructive periodontal diseases.

76 subjects with prior evidence of destructive periodontal diseases were monitored clinically and immunologically every 2 months for up to 5 years. Clinical parameters measured included bleeding on probing, gingival redness, plaque accumulation, suppuration, pocket depth and attachment level. Blood samples were taken by venipuncture and serum antibody levels to a series of 18 subgingival species determined. 33 of these subjects showed evidence of active disease during the monitoring period, based on changes in attachment level measurements assessed using the tolerance method of analysis. Mean attachment loss in these 33 subjects varied from 1.4 mm to 9.0 (median value 3.4 mm) and subjects whose mean attachment level was above the median showed a higher % of pockets greater than 3 mm and more suppuration. Severity of gingival inflammation related poorly to mean attachment loss. Subgingival plaque samples were taken from the active site(s) and from control sites of equal pocket depth and attachment loss in the same active disease subjects, prior to therapy, for predominant cultivable microbiota studies. 50 randomly selected isolates were identified from each sample. Predominant cultivable species in 170 pretreatment active and inactive sites combined (8500 isolates) were enumerated. The most frequently detected species were F. nucleatum (112 sites) and S. intermedius (106 sites), although the predominant species in the samples from each subject differed. The distribution of putative pathogens differed among subjects. For example, A. actinomycetemcomitans was found in 21 samples in 11 subjects and B. forsythus was found in 18 samples from 10 individuals. Antibody response patterns to the 18 subgingival species also varied among subjects.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of modified Widman flap surgery and systemic tetracycline on the subgingival microbiota of periodontal lesions.

33 subjects with evidence of active destructive periodontal disease were treated by modified Widman flap surgery and systemic tetracycline (1 g/day for 21 days). Subgingival plaque samples were taken from 41 sites in 12 of these subjects before and 6 months after therapy for predominant cultivable microbiota studies. Mean pocket depth and attachment levels in the 41 sampled sites were 7.1 +/- 2.9 mm and 7.7 +/- 3.2 mm prior to therapy and 4.8 +/- 2.3 mm and 6.2 +/- 3.4 mm after therapy. B. melaninogenicus and V. parvula were more frequently detected in samples taken after therapy, while S. intermedius, S. morbillorum, S. uberis and W. recta were less frequently detected after therapy. A. actinomycetemcomitans were detected in 7 sites pretherapy and 1 site post therapy. The frequency of detection of B. gingivalis and B. intermedius was virtually unchanged. The mean levels of the Actinomyces sp., A. actinomycetemcomitans, B. gingivalis, B. intermedius, S. morbillorum, S. uberis and W. recta were decreased after therapy, while the mean levels of B. melaninogenicus, S. mitis, S. sanguis II and V. parvula were increased after therapy. V. parvula showed the greatest increase to 8.2% of the microbiota. In the second phase of the study, subgingival plaque samples from 94 sites in the 33 treated subjects were analyzed by predominant cultivable techniques. As a result of therapy, 24 sites exhibited attachment loss greater than 2 mm, 23 sites exhibited "gain" greater than 2 mm and the remaining 47 sites were considered to be unchanging.(ABSTRACT TRUNCATED AT 250 WORDS)

Difficulties encountered in the search for the etiologic agents of destructive periodontal diseases.

The present paper outlines some of the difficulties encountered in the search for the etiologic agents of destructive periodontal diseases. These include technical problems such as acquiring an appropriate microbial sample, as well as difficulties in the dispersion, cultivation and identification of isolates in that sample. Many of these difficulties are currently being successfully addressed. A second set of problems is more conceptual in nature. These include difficulties in distinguishing between periodontal diseases and determining the state of activity of periodontal lesions. In addition, complexes of organisms and/or sequences of species may be involved in the progress of lesions. A further problem is encountered in attempting to distinguish overgrowths of opportunistic species from increases in proportions of true pathogens. Finally, it appears likely that different infections occur at the same time in a single oral cavity. The technical and conceptual difficulties eventually filter down to the data analytical step and present numerous problems to the analyst. With all of these difficulties in mind, it is not surprising that the etiologic agents of destructive periodontal diseases are not clearly defined. However, improvement in technological assessments of the microbiota and clinical evaluation of the disease should permit reasoned approaches to be taken. The delineation of the etiologic agents of destructive periodontal diseases will be, of necessity, a multistage iterative process. Etiologic agents will be suggested by predominant cultivable studies and hypotheses concerning subsets of these agents tested using more specific procedures such as selective media, immunofluorescent techniques or DNA probes.(ABSTRACT TRUNCATED AT 250 WORDS)

Development of a broth medium for Bacteroides forsythus.

Bacteriodes forsythus has been implicated as a possible pathogen in certain destructive periodontal diseases. This species has been extremely difficult to grow in broth, thus hampering its characterization and identification. A broth medium was developed consisting of 30 g of phytone, 10 g of NaCl, and 1 g of filter-sterilized L-cysteine per liter. The medium supported the growth of 23 fresh isolates of B. forsythus from six subjects to optical densities averaging 0.71 +/- 0.20 (standard deviation) at 550 nm after 48 to 72 h of anaerobic incubation.

Gram negative species associated with active destructive periodontal lesions.

Apical subgingival plaque samples were taken from 19 subjects exhibiting active destructive periodontal disease. The predominant cultivable Gram negative species from 50 active sites were compared to 69 inactive sites of comparable pocket depth and attachment level loss. Active disease sites were chosen which showed a significant loss of attachment within a two-month interval. Proportions of Gram negative rods were higher in active periodontal disease sites than in inactive sites. Species which were found to be significantly elevated only in active sites were Bacteroides intermedius, "fusiform" Bacteroides, Actinobacillus actinomycetemcomitans and Wolinella recta. Fusobacterium nucleatum, Capnocytophaga gingivalis and Eikenella corrodens were found in significantly increased proportions in active sites of some subjects and inactive sites of others.

Chemically defined medium for oral microorganisms.

We formulated a chemically defined medium consisting of 14 inorganic salts, 23 amino acids, 23 vitamins and other factors, seven purines and pyrimidines, and glucose which would successfully support the growth of a wide variety of oral microorganisms. Of 204 oral isolates representing 20 genera and 60 species, 197 maintained viability through six serial transfers.

Comparative in vitro activity of sanguinarine against oral microbial isolates.

MICs of sanguinarine were determined for 52 oral reference strains and 129 fresh isolates from human dental plaque. Sanguinarine was found to completely inhibit the growth of 98% of the isolates at a concentration of 16 micrograms/ml.

Antibiotic sensitivity patterns of Campylobacter jejuni/coli isolated from laboratory animals and pets.

Fifty-four strains of Campylobacter jejuni/coli isolated from a variety of species of laboratory animals as well as pet dogs were tested by an agar dilution technique for susceptibility to each of 12 antimicrobial agents. Gentamicin and furazolidone were the most active of the drugs examined. The strains tested frequently were sensitive to two other aminoglycoside antibiotics, neomycin and kanamycin. Erythromycin also was effective at levels achievable in serum except in three strains which were resistant. Doxycycline and chloramphenicol were active against most strains, with 51 (96%) being susceptible to 8 micrograms/ml. The minimal inhibitory concentrations were high for penicillin, ampicillin, tetracycline, metronidazole, and sulfadimethoxine.

Semiautomated technique for identification of subgingival isolates.

A semiautomated approach for the characterization of subgingival bacterial isolates which economizes in media preparation, inoculation, reading, recording, and interpretation of results was tested. Test ingredients were added to a basal medium consisting of Mycoplasma broth supplemented with 5 micrograms of hemin, 0.5 mg of NaHCO3, and 0.5 mg of L-cysteine per ml. Sterile test media were aseptically dispensed into wells of sterile microtiter plates with a MIC 2000 dispenser. Inocula were grown in broth or scraped from agar plates, dispersed, and inoculated with a MIC 2000 inoculator. After 2 to 4 days of incubation, the optical density of growth was determined with an Artek 210 vertical beam reader at 580 nm and stored on a floppy disk. Reagents were added to each well, and the changes in optical density were determined. Thresholds for positive reactions were determined after extensive preliminary studies for each test. The tests were run in duplicate on each plate and interpreted with an Artek vertical beam reader. Tests that were run in this system included: fermentation of carbohydrates, decarboxylase reactions, reduction of nitrate and nitrite, ammonia production, hydrolysis of esculin, growth in the presence of inhibitory or stimulatory substances, and indole production. Approximately 80% of all isolates from subgingival samples could be characterized by this technique. Comparisons were made between the semiautomated and conventional identification techniques. Overall reproducibility of 2,980 strains by the semiautomated and conventional techniques were 95 and 90%, respectively. There was an 86% similarity of results by the semiautomated and conventional methods. The semiautomated technique was more rapid, less expensive, and as reproducible as the conventional method of identification.

Antibiotic levels in infected and sterile subcutaneous abscesses in mice.

The pharmacokinetic properties of 10 antimicrobial agents were examined in sterile and infected encapsulated subcutaneous abscesses in mice. The inoculum for sterile abscesses was autoclaved cecal contents; that for infected abscesses was autoclaved cecal contents combined with Bacteroides fragilis. The antimicrobial agents examined were rosaramicin, clindamycin, chloramphenicol, metronidazole, and six beta-lactam antibiotics. All antimicrobial agents entered abscesses, produced peak levels of biological activity that were somewhat delayed in comparison to serum levels, and were present in negligible levels 8 hr after administration. The highest concentration in abscesses was achieved with rosaramicin and clindamycin, with peak levels of 43%--63% of the peak serum level. Peak levels of other antimicrobial agents in sterile abscesses were 13%--27% of the peak serum level. Levels of biologically active during were significantly lower in infected abscesses than in sterile abscesses for antimicrobial agents that are inactivated by B. fragilis beta-lactamase.