Tetracycline fiber therapy monitored by DNA probe and cultural methods.

Oligonucleotide DNA probe and selective cultural methods were compared in their ability to monitor 6 putative periodontal pathogens in a study evaluating local tetracycline fiber therapy. Subgingival plaque was sampled from 4 sites in each of 20 subjects. Samples were taken before and after therapy from sites assigned to the following test groups: tetracycline (TC) fiber, scaling and root planing, control fiber, and untreated. Each sample was analyzed by both DNA probe and cultural methods. Total anaerobic cultivable counts, Porphyromonas (Bacteroides) gingivalis and Prevotella intermedia (Bacteroides intermedius) were enumerated on nonselective blood agar. Actinobacillus actinomycetemcomitans, Eikenella corrodens, Fusobacterium nucleatum and Wolinella recta were isolated on selective media. TC fiber therapy and scaling reduced total cultivable counts from an initial value of 1 x 10(7) to approximately 2 x 10(5) following therapy. Total counts at untreated sites and at sites with control fibers did not change from baseline. A. actinomycetemcomitans and E. corrodens were detected more frequently by the cultural method; the other monitored species were detected more frequently by DNA probes than by the cultural methods. Agreements between methods were: 77.2% for A. actinomycetemcomitans; 72.2% for P. intermedia; 75.6% for E. corrodens; 39.4% for F. nucleatum; 35.6% for P. gingivalis; and 68.9% for W. recta. Limitations of the selective cultural methods used probably contributed to the discrepancies for P. gingivalis and F. nucleatum. DNA probe and cultural methods indicated comparable levels of suppression of the monitored species following TC fiber therapy and scaling. The microbiota of control fiber and untreated sites did not appear to be significantly altered by either method.

Purification and characterization of a 65-kilodalton diisopropylfluorophosphate-binding protein in the outer membrane of Fusobacterium nucleatum Fev1.

A 65-kilodalton protein was identified in the outer membrane of Fusobacterium nucleatum Fev1 by SDS-PAGE. The relative amount of the protein varied during growth, being greatest in stationary phase. The protein was radio-labeled by [125I]-lactoperoxidase treatment of live cells and was only partially extractable with 2% sodium dodecylsulfate (SDS) at room temperature, and therefore assumed to be both exposed to the cell surface and peptidoglycan associated. In intact cells the protein bound diisopropylfluorophosphate (DFP), indicating that it may be a serine protease. DFP-binding activity depended apparently on proper localization of the proteins in the membrane. Several methods were tried in attempts to purify the 65-kilodalton protein; the method that gave best results was preparative SDS-polyacrylamide gel electrophoresis. The isoelectric point was determined to about pH 5. Amino acid composition analysis showed that the 65-kilodalton protein possesses an excess of hydrophilic over hydrophobic residues, polarity index 52%. The N-terminal end of the protein was apparently blocked.

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.

Purification and partial characterization of a major outer-membrane protein of Fusobacterium nucleatum.

The major outer-membrane proteins of 40-41 kDa were identified by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) in Fusobacterium nucleatum strains ATCC 10953, ATCC 25586, F3, F6 and Fev1. The proteins were purified by preparative gel electrophoresis. Their behaviour in gel filtration and gel electrophoresis, their sensitivity to proteolytic enzymes, and their amino acid composition were investigated. The purified proteins were partly sequenced from the N-terminal end. A 36.5 kDa portion was protected against extrinsic proteolytic (trypsin, chymotrypsin or pronase) digestion of whole cells. This polypeptide was isolated and partially sequenced from the N-terminal end. From these data and data from extrinsic iodination it was concluded that the N-terminal end of the protein is probably exposed on the surface of the cell. A database search revealed amino acid sequence similarity in an Ala-Pro-rich region of outer-membrane protein A (OmpA) in other Gram-negative bacteria.

A pyrolysis mass spectrometry study of fusobacteria.

Strains of fusobacteria (143) were examined by pyrolysis mass spectrometry (Py-MS) with a Horizon Instruments PYMS 200X. Fusobacterium necrogenes, F. necrophorum, F. nucleatum, F. mortiferum, F. varium, F. gonidiaformans, F. naviforme, F. russii and Leptotrichia buccalis were discriminated. Strains of fusobacteria isolated from tropical ulcers, although similar to F. mortiferum in conventional tests, were discriminated from each of these species in Py-MS. Identification of 416 spectra to species level agreed with conventional bacteriological methods in 91.8% of cases, was equivocal in 3.4% and disagreed in 4.8%. Classification based on pyrolysis data resolved groups largely corresponding to the recognised species. However, F. nucleatum strains were divided between two distinct groups. The tropical ulcer strains were resolved as a distinct homogeneous group. Py-MS is a rapid, inexpensive and convenient procedure for characterisation of bacteria, with the capacity for a high throughput of samples, although the initial cost of the apparatus is high.

Isolation of a corncob (coaggregation) receptor polypeptide from Fusobacterium nucleatum.

Corncobs, which are distinct morphological units formed by the ordered coaggregation of a filamentous microorganism and streptococci, can be made in vitro by using oral strains of Fusobacterium nucleatum and Streptococcus sanguis. Previous studies have shown that strains of F. nucleatum contain one of at least two different types of corncob receptor. The objective of this study was to isolate the receptor from F. nucleatum ATCC 10953 as the first step in the elucidation of the molecular basis of corncob formation. The cell envelope fraction from this bacterium was treated with trypsin, delipidated with chloroform-methanol, and subjected to ion-exchange chromatography. A single polypeptide (apparent Mr, 39,500), which was eluted from the column with 0.5 M sodium chloride and extracted with dodecyltrimethylammonium bromide to remove contaminating lipopolysaccharide, inhibited corncob formation between strain ATCC 10953 and S. sanguis CC5A. Similarly derived cell fractions from either F. nucleatum FDC 364 or Fusobacterium necrophorum failed to effect coaggregation in the inhibition assay. Amino acid analysis of the polypeptide showed a moderately hydrophobic character (polarity index, 41) and 11% basic residues. Antiserum made against the purified polypeptide agglutinated F. nucleatum ATCC 10953, neutralized the ability of this bacterium to form corncobs, and agglutinated whole cells of S. sanguis CC5A that were precoated with the receptor polypeptide. The identification and isolation of this receptor should greatly enhance our ability to define some of the complex intergeneric coaggregation mechanisms that are thought to occur in the human oral cavity.

Immunobiological properties of lipopolysaccharides isolated from Fusobacterium nucleatum and F. necrophorum.

Lipopolysaccharides (LPSs) were isolated from Fusobacterium nucleatum ATCC 10953 and F. necrophorum ATCC 25286 by the hot phenol/water procedure. F. nucleatum LPS was composed of 16% (w/w) carbohydrate, 10% (w/w) hexosamine and 40% (w/w) fatty acid, while F. necrophorum LPS was composed of 26% (w/w) carbohydrate, 12% (w/w) hexosamine and 28% (w/w) fatty acid. These LPS preparations induced mitogenic responses in spleen cells of BALB/c, BALB/c (nu/nu) and C3H/HeN mice, and these responses were suppressed by the addition of polymyxin B. The preparations also induced the polyclonal responses of C3H/HeN spleen cells. In addition, enhanced glucose utilization and interleukin-1 production by murine peritoneal macrophages were demonstrated. Neither spleen cells nor macrophages from the 'LPS-nonresponsive' C3H/HeJ mouse were activated by LPSs from the Fusobacterium species.

Immunobiological activities of a porin fraction isolated from Fusobacterium nucleatum ATCC 10953.

From Fusobacterium nucleatum ATCC 10953 cell envelope fraction whose inner membranes had been removed by treatment with sodium N-lauroyl sarcosinate, an outer membrane protein (37,000 Mr in a native state) was prepared by extraction with lithium dodecyl sulfate. The protein thus obtained showed distinct porin activity, namely, the ability to form hydrophilic diffusion pores by incorporation into the artificial liposome membrane. The porin fraction exhibited strong immunobiological activities in the in vitro assays: B-cell mitogenicity and polyclonal B-cell activation on murine splenocytes, stimulatory effects on guinea pig peritoneal macrophages, and enhancement of the migration of human blood monocytes. The porin fraction also exhibited immunoadjuvant activity to increase the antibody production against sheep erythrocytes in the spleen of mice that were immunized by sheep erythrocytes with porin. Although chemical analyses revealed that the test porin fraction contained a considerable amount of lipopolysaccharide (LPS) (around 12% of the fraction), the studies with LPS-nonresponding C3H/HeJ mice and on the inhibitory effects of polymyxin B strongly suggest that most of the above bioactivities are due to porin protein itself, not to coexistent LPS in the porin fraction.

Chemical composition and immunobiological activities of sodium dodecyl sulphate extracts from the cell envelopes of Actinobacillus actinomycetemcomitans, Bacteroides gingivalis and Fusobacterium nucleatum.

The chemical composition and immunobiological activities in vivo and in vitro of sodium dodecyl sulphate extracts (SDS-SE) derived from periodontopathic bacteria (three strains of Actinobacillus actinomycetemcomitans, two strains of Bacteroides gingivalis, and one strain of Fusobacterium nucleatum) were investigated. The main components of SDS-SE were protein and lipid, with negligible amounts of peptidoglycan and lipopolysaccharide. Immunopotentiating activity was detected in both delayed-type hypersensitivity and antibody formation against the elicitation of a protein antigen with the SDS-SE preparations of A. actinomycetemcomitans ATCC 29524 and B. gingivalis 381 and 1021. On the other hand the SDS-SE of A. actinomycetemcomitans ATCC 29522 enhanced only the induction of a delayed-type hypersensitivity response. All the SDS-SE preparations had mitogenic activity to splenocytes from BALB/c nu/nu, C3H/HeN and C3H/HeJ mice. Migration-stimulating activity for human peripheral blood monocytes was detected especially in the SDS-SE preparations of A. actinomycetemcomitans ATCC 29524 and Y4. All of the SDS-SE samples inhibited [3H]thymidine uptake in human gingival fibroblasts and caused degradation of the cells. The results suggest that the cell membrane components extractable with sodium dodecyl sulphate from periodontopathic bacteria are involved in the pathogenesis of periodontal disease.

Studies on the purification of the leucocidin of Fusobacterium necrophorum and its neutralization by specific antisera.

Leucocidin from several strains of Fusobacterium necrophorum was partially purified by gel filtration on Fractogel HW55 (F), the majority of the activity being present in the 50 ml of filtrate collected after 1.1 void volumes had passed through the column (termed Fraction 1, or #1). The material also contained lipopolysaccharide in 12.5% SDS-PAGE gels run under reducing conditions, but the protein did not migrate into 7.5% PAGE gels run under non-reducing conditions. Rabbit and bovine antisera to the leucocidin possessed antibodies against antigens in concentrated, washed culture supernates from toxigenic F. necrophorum and neutralized the leucocidal activity of such supernates. Absorption of the antisera with homologous, washed F. necrophorum cells reduced ELISA antibody titres by greater than 50%, but decreased neutralization titres by 15%. Absorbed rabbit IgG anti-#1 precipitated a single rocket in crossed immunoelectrophoresis and identified two proteins, of molecular weights (M.W.) 14 000 and 13 000, and 1 protein of M.W. 13 500 in immunoblots from toxigenic and non-toxigenic strains, respectively. An additional protein of M.E. 103 000 was present after SDS-PAGE separation of supernates from toxigenic but not non-toxigenic F. necrophorum and was not present in whole cell components. It was considered that the leucocidin may be present in a dimeric form in culture supernates from toxigenic strains. Antisera to leucocidins from several strains of F. necrophorum exhibited variable neutralization titres against leucocidins from heterologous bacteria.

Outer membrane proteins of Fusobacterium nucleatum Fev1.

Outer membrane enriched material from six strains of Fusobacterium nucleatum was analysed by SDS-PAGE. The protein profiles of all the strains were dominated by proteins with molecular masses of about 40 kDa, and a very high degree of homology in relation to apparent molecular masses was observed. In all strains except Fev1, one of the most dominant proteins exhibited heat modifiable properties, having an apparent molecular mass of about 38 kDa and 42 kDa when heated in SDS at 50 and 100 degrees C, respectively. None of the proteins of the outer membrane of F. nucleatum Fev1 demonstrated such heat modifiable properties. The 40 kDa protein, and several other proteins, appear to be both exposed on the cell surface and peptidoglycan associated.

The relationship between Fusobacterium species and other flora in mixed infection.

Mixed infections with three Fusobacterium species and seven other bacterial species were studied in a subcutaneous abscess model in mice. Fifteen Fusobacterium isolates (eight F. nucleatum, four F. necrophorum, and three F. varium) and one isolate each of Bacteroides fragilis, B. asaccharolyticus, Staphylococcus aureus, Group A beta-haemolytic streptococcus, Escherichia coli, Klebsiella pneumoniae and Pseudomonas aeruginosa were studied. Electronmicrographs showed the presence of a thin mucopolysaccharide wall before and after inoculation into mice in 12 isolates which included all of 11 Fusobacterium isolates that induced subcutaneous abscesses. After co-inoculation of Fusobacterium isolates with other species and selective therapy with antimicrobial agents, S. aureus and K. pneumoniae were found to be of equal or greater importance in abscess induction than were Fusobacterium isolates, while Fusobacterium isolates were found to be more important than Group A streptococci and E. coli. Mutual enhancement of the numbers of organisms in mixed infections was observed with Fusobacterium spp. and K. pneumoniae, P. aeruginosa or Bacteroides spp. Suppression of Fusobacterium spp. was noticed only when they were co-inoculated with Group A streptococci. The additive or synergistic capabilities of Fusobacterium species highlighted their potential pathogenicity in infection.

Fatty acid composition and Shwartzman activity of lipopolysaccharides from oral bacteria.

The composition and the nature of the linkage of fatty acids and the Shwartzman activity of lipopolysaccharide (LPS) preparations derived from oral gram-negative bacteria including Bacteroides gingivalis, Bacteroides loesheii, Eikenella corrodens, Fusobacterium nucleatum, and Actinobacillus actinomycetemcomitans were examined. 3-Hydroxylated and nonhydroxy fatty acids of various chain lengths were found in all of the LPS preparations. All nonhydroxy fatty acids were found to be ester-bound, and part of the 3-hydroxy fatty acids in the LPS of B. gingivalis, E. corrodens, F. nucleatum, and A. actinomycetemcomitans were shown to be involved in ester linkage. It was also suggested that the hydroxy group of the ester-bound 3-hydroxy fatty acid of the LPS of F. nucleatum and A. actinomycetemcomitans is at least partly substituted by another fatty acid, but in the LPS of B. gingivalis and E. corrodens it is not. The main amide-linked fatty acid of the LPS of B. gingivalis, E. corrodens, F. nucleatum, and A. actinomycetemcomitans was 3-hydroxyheptadecanoic, 3-hydroxydodecanoic, 3-hydroxyhexadecanoic, and 3-hydroxytetradecanoic acid, respectively. The results of the Shwartzman assay showed that the E. corrodens LPS was the most active among the preparations tested, and that the Shwartzman toxicity of Bacteroides LPS is extremely low.

Nature and location of amide-bound (R)-3-acyloxyacyl groups in lipid A of lipopolysaccharides from various gram-negative bacteria.

It has previously been demonstrated [Eur. J. Biochem. 124, 191-198 (1982) and 137, 15-22 (1983)] that the lipid A component of Salmonella and Proteus lipopolysaccharides contains amide-linked (R)-3-acyloxyacyl residues. In the present study lipid A of other gram-negative bacteria was analysed for the presence of amide-bound 3-acyloxyacyl residues. It was found that such residues are constituents of all lipid A tested (Agrobacterium tumefaciens, Chromobacterium violaceum, Pseudomonas aeruginosa, Xanthomonas sinensis, Bacteroides fragilis, Vibrio cholerae, Fusobacterium nucleatum, Rhodospirillum tenue, Acinetobacter calcoaceticus, and Escherichia coli). Amide-linked (R)-3-acyloxyacyl groups, therefore, represent common and ubiquitous structural elements of bacterial lipid A. The composition of 3-acyloxyacyl groups differed considerably among different bacteria. As amide-bound (R)-3-hydroxy fatty acids straight chain and isobranched acyl groups with 10-17 carbon atoms were identified. The most frequently encountered fatty acids, substituting the 3-hydroxyl group of 3-hydroxy fatty acids, were nonhydroxylated straight chain and isobranched acyl residues with 10-17 carbon atoms as well as (S)-2-hydroxy fatty acids with 12 carbon atoms. In some cases, using laser desorption mass spectrometry, the distribution of 3-acyloxyacyl residues over the two available glucosamine amino groups of the lipid A backbone was investigated.