The mechanism of Eikenella corrodens adherence to human buccal epithelial cells in vitro was studied. Initial experiments to determine the optimal conditions for adherence of E. corrodens to buccal epithelial cells showed that adherence was dependent on time, temperature, bacterial concentration, and pH. Different strains of E. corrodens varied in their ability to adhere, and strain 1073 showed the greatest ability in adherence. Strain 1073 was selected for studies of adherence mechanisms. Trypsin treatment or heating (100 degrees C, 10 min) of the bacterial cells abolished their capacity to adhere to buccal epithelial cells. Treatment of buccal epithelial cells with trypsin also abolished adherence of E. corrodens 1073, whereas neuraminidase treatment of buccal epithelial cells enhanced the adherence. The adherence was inhibited by ethylenediaminetetraacetic acid and restored by adding Ca2+. The adherence was remarkably inhibited by sugars containing D-galactose and n-acetyl-D-galactosamine. Treatment of neuraminidase-treated epithelial cells with sodium metaperiodate or alpha- and beta-galactosidase did not decrease the adherence. These data suggest that adherence of E. corrodens 1073 to human buccal epithelial cells may require the interaction of lectin-like proteins on the bacterial surface with galactose-like receptors on the surface of epithelial cells.

Association constants for the binding of methyl alpha-D-galactopyranoside (methyl alpha-D-Galp) and methyl 2-acetamido-2-deoxy-alpha-D-galactopyranoside (methyl alpha-D-GalNAcp) to three Bandeiraea simplicifolia isolectins (A4, A2B2, B4) were determined by equilibrium dialysis and fluorescence enhancement measurements. The a and B subunits appear to have approximately the same Kassoc for methyl alpha-D-Galp: 1.45 X 10(4), 1.98 X 10(4), and 2.06 X 10(4) M-1 for A4, A2B2, and B4, respectively, as determined by equilibrium dialysis. Fluorescence enhancement measurements on B4 gave an association constant of 2.07 X 10(4) M-1 for methyl alpha-D-Galp and 1.87 X 10(3) M-1 for methyl beta-D-galp. By equilibrium dialysis, we were able to detect 3.3 (theory, 4.0) methyl alpha-D-GalNAcp binding sites for A4 (Kassoc = 1.87 X 10(5) M-1), 1.9 for A2B2 (Kassoc = 1.19 X 10(5) M-1), and were unable to detect any methyl alpha-D-GalNAcp binding sites for B4. However, four very weak methyl alpha-D-GalNAc binding sites for B4 were detected by fluorescence enhancement measurement (Kassoc = 1.26 X 10(2) M-1). Thus, the A subunit has an affinity for methyl alpha-D-GalNAc 3 orders of magnitude greater than the B subunit. Precipitation and hapten inhibition data are in accord with these binding measurements. Toward guaran and type B blood group substance, all isolectins precipitated the same amount of biopolymer. However, AB3, A2B2, and A3B, which are mono-, di-, and trivalent for alpha-D-GalNAcp, were differentially precipitated by type A blood group substance which contains alpha-D-GalNAcp-end groups. A3B precipitated the most, A2B2 less, and AB3 no type A substance. These isolectins should prove useful in studies evaluating the effect of valence on lectin-cell interaction.

The conformational model derived by the HSEA calculation method was used to interpret the n.m.r. data for solutions of oligosaccharides corresponding to the Salmonella serogroups A, B, and D1 antigenic determinants. The favored conformer, derived by calculation, accounted for the observed, chemical-shift changes and accurately predicted the existence and magnitude of inter-ring proton n.O.e.'s. Extensive proton-density and compression of proton, Van der Waals radii were correlated with deshielding of specific proton-resonances. The model of lipopolysaccharide conformation accounts for the known antigenic properties of Salmonella O-antigens.

The preferred conformation of the tetrasaccharide repeating units of Salmonella Serogroups A, B, and D1 have been calculated. The semiempirical calculations used the Hard-Sphere, Exo-Anomeric (HSEA) approach to derive a conformational model, which could be used to assist the interpretation of conformations significantly populated in aqueous solution. The calculated model was extended to include a pentasaccharide repeating unit bearing an alpha-D-glucopyranose-branch point. The 3,6-dideoxyhexose to D-mannose linkage was shown to possess a steep energy surface with a minimum, which results in good exposure of the dideoxyhexose O-2 and O-4 atoms. Stereochemical changes involving the equatorial or axial disposition of these atoms are the distinguishing structural features of the A, B, and D1 serogroups. A lipophilic surface involving the 6-deoxy groups of the dideoxy-D-hexose and L-rhamnose residues was identified, and the possible implications of these features in antigenic determinants is discussed. The preferred conformation predicted by the HSEA method correlates with the known antigenic specificities of polysaccharides belonging to these Salmonella serogroups.

AIMS To investigate the effects of the combined application of an N-acyl homoserine lactone (HSL) analog and antibiotics on biofilms of Porphyromonas gingivalis, a major pathogen of periodontal disease. METHODS AND RESULTS Antibiotics used were cefuroxime, ofloxacin and minocycline. A flow-cell model was used for biofilm formation. Samples were divided into four groups: control, analog-treated, antibiotic-treated and combined application groups. Biofilm cell survival was determined using adenosine triphosphate (ATP) bioluminescence and confocal laser microscopy (CLSM). In the combined application group, the ATP count in biofilm cells was significantly decreased compared with the antibiotic-treated group (Games-Howell test, P < 0·05). A combination of cefuroxime and the analog was most effective against the P. gingivalis biofilm. CLSM observations revealed that the proportion of dead cells was highest in the combined application group. CONCLUSIONS The combined application of the N-acyl HSL analog and antibiotics was effective at reducing the viability of P. gingivalis cells in biofilms. SIGNIFICANCE AND IMPACT OF THE STUDY The combined application of the N-acyl HSL analog and antibiotics may be successful for eradicating infections involving bacterial biofilms, such as periodontitis.

Antibiotic resistance of biofilm-grown bacteria contributes to chronic infections such as marginal and periapical periodontitis, which are strongly associated with Porphyromonas gingivalis. Concurrent azithromycin (AZM) administration and mechanical debridement improve the clinical parameters of periodontal tissue in situ. We examined the in vitro efficacy of AZM against P. gingivalis biofilms. The susceptibility of adherent P. gingivalis strains 381, HW24D1, 6/26 and W83 to AZM, erythromycin (ERY), ampicillin (AMP), ofloxacin (OFX) and gentamicin (GEN) were investigated using a static model. The optical densities of adherent P. gingivalis cells were significantly decreased by using AZM and ERY at sub-MIC compared with controls in all the strains tested, except for the effect of ERY on strain W83. AMP and OFX inhibited P. gingivalis adherent cells at over their MICs, and GEN showed no inhibition in the static model. The effects of AZM and ERY against biofilm cells were investigated using a flow cell model. The ATP levels of P. gingivalis biofilms were significantly decreased by AZM at concentrations below the sub-MICshowever, ERY was not effective for inhibition of P. gingivalis biofilm cells at their sub-MICs. Furthermore, decreased density of P. gingivalis biofilms was observed three-dimensionally with sub-MIC AZM, using confocal laser scanning microscopy. These findings suggest that AZM is effective against P. gingivalis biofilms at sub-MIC levels, and could have future clinical application for oral biofilm infections such as chronic marginal and periapical periodontitis.

HASH(0xfe09cb0)