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.

Outer membrane protein and lipopolysaccharide heterogeneity among Eikenella corrodens isolates.

Outer membrane protein (OMP) and lipopolysaccharide (LPS) phenotypic diversity among 27 oral and extraoral strains of Eikenella corrodens was assessed by SDS-PAGE. Each strain exhibited one to three major protein bands in the 35- to 41.5-kDa range and one or two protein bands of lesser density in the 24.5- to 28-kDa range. Eleven OMP patterns were distinguished among the strains. While oral strains obtained from periodontally healthy and diseased subjects exhibited diverse OMP patterns, five of six strains from extraoral sites of infection expressed an identical OMP pattern. Comparison of the electrophoretic mobilities of LPS from these same strains revealed that E. corrodens LPS consists primarily of low apparent molecular mass forms. Sixteen different LPS phenotypes were differentiated among the strains, with no apparent correlation between LPS phenotype and clinical setting. Strains expressing the same OMP pattern frequently expressed variable LPS phenotypes and vice versa. Analysis of OMP or LPS pattern by SDS-PAGE may be useful in taxonomic and epidemiologic studies of E. corrodens. Additional studies assessing the potential influence of OMP composition on invasiveness of this organism appear warranted.

Cellular fatty acid composition of Kingella species, Cardiobacterium hominis, and Eikenella corrodens.

We determined the cellular fatty acid composition of reference strains and clinical isolates of each of the three Kingella species, Cardiobacterium hominis, and Eikenella corrodens by using capillary gas chromatography. Kingella denitrificans and Kingella kingae contained myristic (14:0) and palmitic (16:0) acids as major acids, whereas cis-vaccenic (18:1 omega 7c) and palmitic acids were the major acids in Kingella indologenes, C. hominis, and E. corrodens. C. hominis differed from the other four species by the absence of 3-hydroxylauric (3-OH-12:0) acid, from K. indologenes by the presence of 3-hydroxypalmitic (3-OH-16:0) acid, and from E. corrodens by the presence of 3-hydroxymyristic (3-OH-14:0) acid. E. corrodens contained a small amount (2%) of myristic acid, while the other four species contained moderate to large amounts (11 to 31%) of this acid.

Some biological activities of Eikenella corrodens major outer membrane proteins.

Major outer membrane proteins of Eikenella corrodens, an organism frequently isolated from patients with periodontal disease, were tested for some biological activities. Mouse peritoneal macrophages, exposed at low concentrations of the above-mentioned proteins (between 0.05 and 5 micrograms/ml), showed evident and marked morphological modifications consisting of increases in the size and vacuolation of the cells. Higher concentrations showed a toxic effect. Low concentrations resulted in a selective release of lysosomal enzymes without any significant release of lactatedehydrogenase, and cytoplasmic marker; while concentrations of 25-50 micrograms/ml, which were toxic in trypan-blue exclusion test, increased LDH release. Eikenella corrodens major proteins increased the platelet aggregation of ADP and thrombin. The residual complement activity of serum samples incubated with various amounts of proteins at 37 degrees C for 30 minutes appeared strongly reduced with respect to controls, thus showing a consumption of the complement components. These results suggested that Eikenella corrodens major proteins may play a role in the development of periodontal lesions.

Characterization of Wolinella spp., Campylobacter concisus, Bacteroides gracilis, and Eikenella corrodens by polyacrylamide gel electrophoresis.

The small asaccharolytic, nonpigmenting gram-negative rods of the human oral cavity are difficult to differentiate from each other. Protein profiles of sonicated cells of Wolinella species, Campylobacter concisus, Bacteroides gracilis, and Eikenella corrodens were obtained by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and visualized with a silver stain. The gels were scanned with a laser densitometer, and the similarity of strains was computed by determining correlation coefficients of normalized densities along the gels. The strains were grouped by cluster analysis of the correlation coefficients. All species were distinct from each other. Several groups were found within E. corrodens. A colored silver stain was found to highlight species differences and appears to be useful in the rapid identification of fresh isolates.

Comparative study of some biological activities of porins extracted from various microorganisms.

The outer membrane of Gram-negative bacteria contains some major proteins, called porins, with particular chemical and physical characteristics which reflect some specialized functions peculiar to the outer membrane. In recent years the biological properties of Salmonella typhimurium SH5014 porins have been the focus of our experimental studies. The aim of the present research was to make a comparative investigation of the biological activities of porins extracted from other microorganisms classified in the Enterobacteriaceae, together with a taxonomically different one, Eikenella corrodens frequently isolated from gum pockets of patients with periodontal disease. Porins from E. coli K12, Proteus mirabilis and Eikenella corrodens were extracted, purified and separated from LPS by means of phenol extraction. The purity of preparation was checked by sodium dodecyl sulphate polyacrylamide gel electrophoresis in slabs. We studied the interactions of these proteins with some cellular and humoral systems within the host. Mouse peritoneal macrophages in the presence of these proteins show morphological and functional modifications, depending upon the type of porins and the concentrations used. The ability of porins to interact with the complement system was investigated in vitro. A pool of sera from 20 blood donors served as a source of human complement. The results obtained with porins of various sources were shown comparatively.

Isolation and characterization of the outer membrane and lipopolysaccharide from Eikenella corrodens.

The chemical composition of the outer membrane fractions (OMFs) of Eikenella corrodens strains 23834 and 470 as well as the strain 23834 lipopolysaccharide (LPS) was determined. The OMFs were obtained by Triton X-100 treatment of the heavier membrane fraction from sucrose density centrifugation of the total membrane fraction. The resulting OMFs of strains 23834 and 470, free of cytoplasmic membrane components, were found to contain 69.6 and 75.0% (wt/wt) protein, 4.8 and 9.2% lipid, 4.6 and 4.7% carbohydrate, and 2.0 and 4.6% muramic acid, respectively. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis both OMFs contained one major peptide determined to be 33,500 daltons for the strain 23834 OMF, and 37,500 daltons for the strain 470 OMF. Analysis of the OMF fatty acids revealed hexadecanoic, hexadecenoic, octadecenoic, and lesser amounts of octadecanoic acids. Transmission electron microscopic examination of the OMFs revealed typical large sheets of membrane. Structures (10 nm in diameter) resembling pores were also evident. The E. corrodens LPS was found to be composed of 34.5% (wt/wt) carbohydrate and 25.0% lipid A. Only minute amounts of 2-keto-3-deoxyoctonate and heptose could be detected. Fatty acid analysis revealed primarily octadecanoic and hexadecanoic acids, with lesser amounts of octadecenoic acid. No hydroxy fatty acids were detected. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis showed the E. corrodens LPS to resemble other smooth-type LPSs. Transmission electron microscopic examination revealed a vesicle-like morphology. The E. corrodens LPS appears not to be a "classical," i.e., enteric, type of LPS.

Biological activity of the slime and endotoxin of the periodontopathic organism Eikenella corrodens.

A partially purified water-soluble slime extract was obtained from two strains of the oral pathogen Eikenella corrodens, designated CS10 A and CS10 B. Endotoxin was also isolated from this organism by the phenol-water extraction procedure. Assays including the local Shwartzman skin reactivity, chicken embryo lethality, Limulus lysate clotting, spleen cell mitogenicity, and immune adjuvancy were used to test the biological properties of these two bacterial extracts. In each of these assay systems the endotoxins of the Eikenella corrodens strains demonstrated the classical endotoxic responses. In contrast, the effect of slime extract in the corresponding assays was either extremely low or absent with the exception of a very strong immunosuppressive effect. Mice treated with slime extract showed a severely reduced immune response to sheep erythrocytes as measured by the hemolytic plaque-forming cell assay.