Isolation and characterization of the cell-surface polysaccharides of Porphyromonas gingivalis ATCC 53978.

The cell-surface carbohydrates of Porphyromonas gingivalis strain ATCC 53978 were isolated and partially characterized. Three separate polysaccharides were found to be present: an extracellular polysaccharide, capsular polysaccharide and lipopolysaccharide. The capsular polysaccharide, which had peculiar, gel-like viscoelastic properties, was found to be comprised of mannuronic acid, glucuronic acid, galacturonic acid, galactose, and 2-acetamido-2-deoxy-D-glucose in relative molar ratios of 0.6:0.9:0.5:0.5:1.0, respectively. The extracellular polysaccharide was found to contain mannose, rhamnose, glucose, galactose, and 2-acetamido-2-deoxy-D-glucose in relative molar ratios of 13.5:1.4:1.0:2.0:1.0, respectively. The lipopolysaccharide was found to contain an O-antigen with a regular tetrasaccharide repeat unit comprised of 4-linked alpha-L-rhamnopyranosyl, 6-linked alpha-D-glucopyranosyl, 3-linked alpha-D-galactopyranosyl, and 4-linked 2-acetamido-2-deoxy-beta-D-glucopyranosyl residues in equimolar proportions.

Localized sinus inflammation in a rabbit sinusitis model induced by Bacteroides fragilis is accompanied by rigorous immune responses.

We evaluated inflammatory and immune responses against Bacteroides fragilis in a rabbit sinusitis model. Bacteroides was inoculated into the left maxillary sinus, and inflammatory (histology, cell number/cytology, lactose dehydrogenase, and apoptosis) and immune responses in the sinus, airway, and peripheral blood (PB) were determined for up to 4 weeks. In the inflamed sinus, the lactose dehydrogenase level was markedly elevated, with neutrophilic infiltration, severe tissue inflammation, and increased apoptosis. Low-grade tissue inflammation was present in the contralateral and sham-operated sinuses, but other parameters remained unchanged, and so did those in the airway and PB in the inoculated rabbits. Serum IgG antibody levels increased rapidly, were highest at 3 weeks, and began to decline at 4 weeks. Cellular immune responses (proliferation and interferon-gamma mRNA expression) against Bacteroides were detected in the PB of all inoculated rabbits. Vigorous immune responses against Bacteroides may have localized but failed to terminate inflammation in the sinus, indicating importance of microenvironmental factors.

Effects of pH, potassium, magnesium, and bacterial growth phase on lysozyme inhibition of glucose fermentation by Streptococcus mutans 10449.

The effects of physiological (saliva and plaque fluid) concentrations of potassium and magnesium and growth phase on lysozyme inhibition of glucose fermentation by S. mutans 10449 were investigated. Glucose fermentations were carried out in a pH-stat at pH 7.0 or 5.5. Cells were at least two times more sensitive to lysozyme in the early-to-middle exponential phase compared with the stationary phase. S. sobrinus 6715 exhibited three-fold greater lysozyme resistance than S. rattus BHT or S. mutans 10449. The concentration of potassium which reduced lysozyme inhibition of S. mutans 10449 fermentation by 50% was 0.2 and 10 mmol/L for stationary and exponential phase cells, respectively. Corresponding values for magnesium were < or = 0.01 and 0.50 mmol/L. Potassium and magnesium exhibited little pH dependence in their reduction of lysozyme inhibition of fermentation by exponential- or stationary-phase S. mutans 10449. The results suggest that: (i) lysozyme interaction with stationary-phase cells involves more non-inhibitory modes than with exponential-phase cells, and (ii) lysozyme may be more effective as an antibacterial agent in saliva than in plaque fluid.

Effect of lysozyme on glucose fermentation, cytoplasmic pH, and intracellular potassium concentrations in Streptococcus mutans 10449.

Several previous findings have suggested that the cationic nature of lysozyme is a major factor in its bactericidal activity. Since a number of cationic proteins or peptides have been reported to cause membrane damage in bacteria, we investigated the effect of lysozyme on glucose fermentation and intracellular pH and K+ in Streptococcus mutans under conditions in which lysis does not occur. Results showed that lysozyme and poly-D-lysine (PDL) cause inhibition of glucose fermentation at pH 5.5 in a dose-dependent manner. Human placental lysozyme and hen egg-white lysozyme exhibited similar inhibitory potency on glucose fermentation. Both lysozyme and PDL caused a marked acidification of the cytoplasm of S. mutans. However, when cytoplasmic pH was examined as a function of fermentation rate, the relationship was similar regardless of the presence or absence of lysozyme or PDL. Therefore, acidification of the cytoplasm appeared to not depend specifically on lysozyme or PDL. In contrast, the same relationship between the profound loss of intracellular K+, when fermenting cells were exposed to either lysozyme or PDL, and the fermentation rate was not exhibited in the controls. These results indicate that lysozyme and PDL specifically affected the ability of the cells to maintain intracellular K+. We concluded that lysozyme and PDL indeed perturb membrane function, perhaps in a selective manner. Furthermore, the similarity in action of lysozyme and the cationic homopolypeptide PDL supports the notion that the cationic property of lysozyme indeed plays a significant role in its antibacterial activity.

Inhibition of bactericidal and bacteriolytic activities of poly-D-lysine and lysozyme by chitotriose and ferric iron.

In a previous report from this laboratory (N. J. Laible and G. R. Germaine, Infect. Immun. 48:720-728, 1985), evidence was presented to suggest that the bactericidal actions of both reduced (i.e., muramidase-inactive) human placental lysozyme and the synthetic cationic homopolymer poly-D-lysine involved the activation of a bacterial endogenous activity that was inhibitable by N,N',N"-triacetylchitotriose (chitotriose). In the present investigation however, we found that the bactericidal and bacteriolytic action of poly-D-lysine could be prevented only by some commercially available chitotriose preparations and not by others. Analysis by physical and chemical methods failed to distinguish protective chitotriose (CTa) and nonprotective chitotriose (CTi) preparations. CTi and CTa preparations displayed equal capacities to competitively inhibit binding of [3H]chitotriose by immobilized lysozyme and were indistinguishable in their abilities to block the lytic activity of lysozyme against Micrococcus lysodeikticus cells. Elemental analysis revealed significantly higher levels of phosphorus, calcium, iron, sodium, manganese, and copper in CTa. Removal of metals from CTa by chelate chromatography completely abolished the poly-D-lysine-protective capacity. Of the metals detected, only ferric iron (5 to 10 microM) mimicked the protective action of CTa. A Fe(III) concentration of 50 microM was required to inhibit lysozyme (5 micrograms/ml). Both Fe(III) and CTa (but not CTi) quantitatively blocked the labeling of poly-D-lysine by fluorescamine, suggesting that the primary amino groups of the lysine residues participate in iron binding. Thus, it appears that the poly-D-lysine-protective capacity of certain chitotriose preparations was due not to the chitotriose itself but to contaminating metal ions which interact directly with the polycationic agent. In contrast, Fe(III) cannot account for inhibition of either the bactericidal or bacteriolytic activity of lysozyme by chitotriose.

Potential role of lysozyme in bactericidal activity of in vitro-acquired salivary pellicle against Streptococcus faecium 9790.

The adherence of Streptococcus faecium 9790 to hydroxyapatite (HA) coated with whole saliva supernatant proteins (S-HA) or parotid fluid proteins was studied. The organism was labeled with [3H]thymidine, and adherence was estimated as the radioactivity remaining associated with the variously coated HA preparations after incubation and removal of unbound microbes by washing the adherence substratum. Adherence was time dependent and saturable, characteristics typical of oral streptococci in this in vitro adherence model system. However, adherence to S-HA, but not bare HA, was decreased 20-fold at 4 degrees C compared with room temperature. Furthermore, adherence at 4 degrees C to S-HA was decreased 20-fold relative to bare HA at 4 degrees C. Adherence to HA coated with parotid fluid proteins also was reduced at 4 degrees C. The magnitude of the temperature dependence and the inhibitory effect at 4 degrees C of whole saliva or parotid fluid pellicles on HA was unexpected. Of several sugars and amino sugars tested, the chitin saccharides, chitotriose, chitobiose, and N-acetylglucosamine caused greater than 90% inhibition of adherence to S-HA. These same saccharides were previously shown to inhibit lysozyme, polylysine, or autolytic lysis of the organism (N. J. Laible and G. R. Germaine, Infect. Immun. 48:720-728, 1985). Examination of unbound and adherent microbes revealed that lysis of the organism occurred during the adherence assays. A strong association (r = 0.83) between the extent of lysis and the extent of adherence was found under a variety of conditions. Depletion of lysozyme from saliva specimens used to coat HA resulted in a greater than 90% decrease in both cell lysis and adherence. Lysis of the microbe appeared dependent upon the presence of the saliva pellicle (coating) on HA, since solutions containing proteins desorbed from HA during mock-adherence incubations possessed lytic activity that was 2- to 10-fold too low to account for the extents of lysis observed with greater than or equal to 10(8) input cells. These results demonstrate the potential antibacterial activity of acquired salivary pellicle on enamel in vivo and the likely role of lysozyme in this activity. The data also serve to caution that this widely used in vitro adherence model will not distinguish whole-cell adherence from the adsorption of radiolabeled DNA released from lysing cells. Several additional controls are suggested that will indicate whether test microbes remain intact or lyse during adherence trials.

Adherence of Streptococcus sanguis to hydroxyapatite coated with lysozyme and lysozyme-supplemented saliva.

The adherence of [3H]thymidine-labeled Streptococcus sanguis strains to bare hydroxyapatite and to hydroxyapatite coated with a range of concentrations of lysozyme, poly-L-lysine, poly-L-glutamic acid, whole saliva supernatant, and combinations of some of the above was studied. Adherence of several strains of S. sanguis to bare hydroxyapatite and saliva-coated hydroxyapatite was compared. Saliva present as a pellicle on the hydroxyapatite inhibited adherence of some strains (903, M-5, 73X11) and stimulated that of others (S35, B-4, 66X49). Strains 903 and S35 were chosen for further study. Adherence of both strains was stimulated up to fivefold by the presence of adsorbed lysozyme or poly-L-lysine on the hydroxyapatite, whereas poly-L-glutamic acid inhibited adherence (80 to 95%). Adherence of strain S35 to hydroxyapatite coated with combinations of saliva and (i) lysozyme, (ii) poly-L-lysine, or (iii) poly-L-glutamic acid was unaffected compared with adherence to hydroxyapatite coated with saliva alone. In contrast, adherence of strain 903 to hydroxyapatite coated with combinations of saliva and either lysozyme or poly-L-lysine was inhibited up to ca. 90% compared with hydroxyapatite coated with saliva alone. Strain 903 was also unaffected by combinations of poly-L-glutamic acid and saliva on the hydroxyapatite. Adherent cells of both strains were completely (greater than 90%) eluted with high-ionic-strength buffer from either bare hydroxyapatite or hydroxyapatite coated with lysozyme alone. Adherent cells of strain S35 were only poorly eluted (25%) from hydroxyapatite coated with either saliva alone or saliva and lysozyme. Strain 903 elution from hydroxyapatite coated with either saliva alone or saliva and lysozyme was essentially complete. These observations were taken to indicate that the two test strains adhered to saliva-coated hydroxyapatite by different mechanisms. Protein-coated hydroxyapatite was shown not to be saturated under the conditions described here. Examination by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the variously supplemented salivary pellicles formed on the hydroxyapatite demonstrated that major changes in salivary protein composition did not occur when lysozyme, poly-L-lysine, or poly-L-glutamic acid was used to supplement saliva. Lysozyme-dependent aggregation of strain 903 was shown not to occur under the conditions of our experiments. We suggest that the basis for stimulation of adherence to hydroxyapatite coated only with lysozyme is an increase in the cationic surface area available for electrostatic adherence of the microorganisms.(ABSTRACT TRUNCATED AT 400 WORDS)

Role of the cell membrane in pH-dependent fluoride inhibition of glucose uptake by Streptococcus mutans.

The effect of pH on the fluoride sensitivity of glucose uptake by whole cells and glucose transport by permeabilized cells of Streptococcus mutans was compared. Whole cells exhibited a marked pH-dependent sensitivity to fluoride over the pH range 7.0 to 5.0. As the pH was decreased, fluoride sensitivity increased. In contrast, no significant effect of pH on the fluoride sensitivity of glucose transport (e.g., phosphorylation) by permeabilized cells energized with 2-phosphoglycerate was noted. The relative effect of pH on the fluoride sensitivity of whole cell glucose uptake and fermentation was similar. These data are consistent with the notion that the cell membrane is impermeable to the fluoride anion and that intracellular accumulation of fluoride depends on translocation of hydrogen fluoride across the membrane.

Effect of endogenous phosphoenolpyruvate potential on fluoride inhibition of glucose uptake by Streptococcus mutans.

The fluoride sensitivity of glucose uptake by whole cell suspensions of Streptococcus mutans was studied. Preincubation of the organism with up to 1 mM glucose markedly reduced the fluoride sensitivity of subsequent glucose uptake at pH 7.0 and 5.5. Glucose preincubation was shown to result in the establishment of a stable pool of three-carbon glycolytic intermediates. On the basis of inhibition studies and thin-layer chromatography of cell extracts, we suggest that 3- and 2-phosphoglycerate are the principal constituents of the pool. Increased concentrations of glucose used in preincubation mixtures was associated with increased pool sizes of the glycolytic intermediates and increased fluoride resistance. Transport of 2-deoxy-D-glucose by permeabilized cells was inhibited by fluoride when 2-phoshoglycerate served as the energy source. Increased concentrations of 2-phosphoglycerate were shown to overcome the fluoride inhibition of transport. The data suggest that establishment of a stable pool of glycolytic intermediates that includes 2-phosphoglycerate (or its progenitors) may contribute significantly to the apparent refractoriness of plaque microbes to fluoride in vivo.

Bactericidal activity of human lysozyme, muramidase-inactive lysozyme, and cationic polypeptides against Streptococcus sanguis and Streptococcus faecalis: inhibition by chitin oligosaccharides.

The basis of the bactericidal activity of human lysozyme against Streptococcus sanguis was studied. Experiments were designed to evaluate the role of lysozyme muramidase activity in its bactericidal potency. Inactivation of the muramidase activity of lysozyme was achieved by reduction of essential disulfides with dithiothreitol (DTT) or by incubation with the chitin oligosaccharides chitotriose and chitobiose. Muramidase-inactive lysozyme, prepared by reduction with DTT, was equal in bactericidal potency to native lysozyme. Solutions of native chicken egg white lysozyme and human lysozyme exhibited equal bactericidal potency yet differed ca. fourfold with respect to lytic (muramidase) activity. The above results suggested that the bactericidal activity of lysozyme is not dependent upon muramidase activity. Chitotriose and chitobiose were found to inhibit both lytic and bactericidal activities of lysozyme. The bactericidal activity of muramidase-inactive lysozyme (reduction with DTT) was also inhibited by chitotriose and chitobiose. Further investigations demonstrated that chitotriose and chitobiose were also potent inhibitors of the bactericidal activity of the cationic homopolypeptides poly-L-arginine and poly-D-lysine. These latter results suggested that the essential bactericidal property of lysozyme was its extreme cationic nature and that some bacterial endogenous activities, inhibitable by chitotriose and chitobiose, were essential for expression of the bactericidal activity of either native or muramidase-inactive lysozyme or of the cationic homopolypeptides. Experiments with Streptococcus faecalis whole cells, cell walls, and crude autolysin preparations implicated endogenous autolytic muramidases as the bacterial targets of chitotriose and chitobiose. The essentially identical responses of S. sanguis and S. faecalis to chitotriose in bactericidal assays with muramidase-inactive lysozyme and polylysine suggested that muramidase-like enzymes exist in S. sanguis and, furthermore, play an essential role in cationic protein-induced loss of viability of the oral microbe.

Promotion of Streptococcus mutans glucose transport by human whole saliva and parotid fluid.

Human saliva and parotid fluid have two effects on glucose uptake by Streptococcus mutans: a reduction in the overall rate of uptake, and the promotion of a biphasic mode of uptake. The former effect had been previously shown to result from lactoperoxidase-mediated inhibition of transport or metabolism or both. The objective of the present study was to uncover the basis of the second effect. Biphasic glucose uptake consisted of a rapid phase of low capacity and short duration (approximately 10 to 15 s) followed by a slower phase of high capacity and long duration (several minutes). The slow phase is typical of cells not exposed to the secretions (control cells). S. mutans BHT cells pretreated with as little as 10 microM glucose for 10 min at 37 degrees C, followed by its removal, subsequently exhibit biphasic glucose uptake typical of saliva- or parotid fluid-treated cells. Since pretreatment of the organism with glucose, whole saliva supernatant, or parotid fluid supported subsequent transport of the nonmetabolized glucose analog, 2-deoxyglucose, we concluded that pretreatments established a relatively stable pool of glycolytic intermediates (i.e., a phosphoenolpyruvate potential). Thin-layer chromatographic analysis of extracts from [14C]glucose-pretreated cells confirmed the presence of a stable pool of triose phosphates. Dialysis experiments indicated that high-molecular-weight substrates in the secretions were readily utilized by the organism to establish a phosphoenolpyruvate potential, especially when the lactoperoxidase system was rendered inactive. A survey of several carbohydrate constituents of salivary glycoproteins revealed that mannose, galactose, and N-acetylglucosamine, in addition to glucose, established phosphoenolpyruvate potentials in the organisms. Inactive substances included, among others, N-acetylgalactosamine and N-acetylneuraminic acid. In a survey of selected amino acids, arginine alone promoted 2-deoxyglucose accumulation by the organism, albeit feebly. Finally, it is argued that the phenomenon of biphasic glucose uptake provides evidence that the rate limiting step in glucose uptake by S. mutans is glucose metabolism and not glucose transport.

Glucose uptake by Streptococcus mutans, Streptococcus mitis, and Actinomyces viscosus in the presence of human saliva.

Glucose uptake was examined by using whole-cell suspensions of Streptococcus mutans (strains BHT, Ingbritt, and GS-5), Streptococcus mitis (strains 9811 and 72x41), and Actinomyces viscosus (strains T6 and WVU626) incubated for up to 90 min in 0 to 82% (vol/vol) human whole salivary supernatant. Glucose uptake by the S. mutans strains was completely inhibited at all saliva concentrations. Dithiothreitol (DTT), present during saliva incubation, prevented saliva inhibition. Glucose uptake was also restored when saliva-inhibited cells were subsequently exposed to DTT. The inclusion of catalase in the saliva incubation mixtures resulted in protection equal to that obtained with DTT. The S. mitis strains were also inhibited by saliva but to a far lesser extent that S. mutans. DTT and catalase also protected S. mitis from saliva inhibition. Both A. viscosus strains were completely refractory to saliva inhibition of glucose uptake. Based on (i) the sensitivity of the catalase-negative streptococci and the resistance of catalase-positive actinomyces to saliva inhibition and (ii) the equal and complete protection to saliva inhibition afforded by DTT and catalase, we conclude that the lactoperoxidase-SCN(-)-H(2)O(2) system in saliva was the only antibacterial system expressed under our experimental conditions. The relative resistance of S. mitis 9811 (compared with S. mutans BHT) to saliva inhibition was shown not to result from poor H(2)O(2) production in either glucose-supplemented buffer or saliva solutions. S. mitis produced inhibitory quantities of H(2)O(2) that equaled or exceeded S. mutans H(2)O(2) accumulation. It is suggested that S. mitis might possess a greater ability to repair lactoperoxidase-mediated damage than does S. mutans. Every organism studied exhibited a saliva concentration-dependent, cell growth-independent stimulation of glucose uptake after 60 to 90 min of incubation. The A. viscosus and S. mitis strains showed saliva stimulation (or stabilization) of glucose uptake with unsupplemented saliva. In the case of S. mutans, saliva stimulation was only observed when DTT was present. The possible role of salivary lactoperoxidase as a modulator of the intraoral site specificities exhibited by S. mutans is discussed.

Adsorption of lysozyme from human whole saliva by Streptococcus sanguis 903 and other oral microorganisms.

Several strains of Streptococcus sanguis, Streptococcus mutans, Streptococcus mitis, Actinomyces viscosus, and Actinomyces naeslundii plus fresh isolates of Streptococcus salivarius were surveyed for their abilities to deplete lysozyme from human-whole-saliva supernatant. Bacteria were incubated in saliva for 60 min at 37 degrees C and then removed by centrifugation, and the recovered supernatant solutions were assayed for lysozyme activity by using whole cells of Micrococcus lysodeikticus as the substrate. Mean lysozyme depletions by bacterial strains varied over a wide (eightfold) range. The greatest mean depletion of lysozyme (60 to 70%) was observed with S. sanguis (biotype I), serotype b of S. mutans, and the fresh S. salivarius isolates. The lowest mean depletion was noted with S. mitis (15%) and biotype II S. sanguis (ca. 30%). The remaining species and strains exhibited an intermediate degree of depletion. In studies with S. sanguis 903, lysozyme was depleted by normal or heated (90 degrees C, 30 min) bacteria and could be recovered from the organism. Furthermore, under appropriate conditions, lysozyme depletion by cells at 0 and 37 degrees C was very similar. On the basis of these observations, we concluded that depletion was due to the adsorption of lysozyme by the organism. With S. sanguis 903, lysozyme adsorption depended on the concentration of bacteria, time of incubation, and the ionic strength of the medium. The extent of adsorption, however, was independent of pH's of 3.9 to 8.3. When a low concentration of S. sanguis 903 was used, lysozyme adsorption reached saturation (4 mug of adsorbed lysozyme per 10(7) cells) at 20 mug of lysozyme added per ml. Salivary lysozyme adsorption by several other oral microorganisms (A. viscosus WVU 626 and WVU 627, S. sanguis 73x11, S. mutans BHT, and S. salivarius NG) was similar to that of S. sanguis 903 in sensitivity to ionic strength. Lysozyme adsorption by S. sanguis 903 from either a buffer solution or a saliva supernatant was more sensitive to ionic strength at 0 than at 37 degrees C. On the basis of results from experiments in saliva versus buffer, we concluded that saliva had no major effect on the extent of lysozyme adsorption by S. sanguis 903 other than providing a source of ionic strength. A comparison of pH and ionic strength effects on lysozyme adsorption by S. sanguis 903 with literature reports of lysozyme lysis of whole cells and hydrolysis of cell walls, peptidoglycan, and (GlcNAc)(4) suggested that adsorption by S. sanguis 903 was more dependent on electrostatic interactions than was lysozyme catalysis. The possibility is discussed that anionic bacterial surface components mediate lysozyme adsorption and temper the potential effects of lysozyme on the microorganisms.

Effect of human saliva on the fluoride sensitivity of glucose uptake by Streptococcus mutans.

The fluoride (F) sensitivity of glucose uptake by whole cell suspensions of streptococcus mutans in the presence and absence of human whole salivary supernatant was studied. It was observed that dithiothreitol (DTT) and other thiols markedly reduced the F sensitivity of cells when saliva (50%, vol/vol) was present during glucose uptake. In the absence of saliva, cells were sensitive to 2 to 2.5 mM F regardless of the presence of thiols. Supplementation of cells in phosphate or tris(hydroxymethyl)aminomethane-hydrochloride buffers with physiological concentrations of calcium or phosphate had no effect on the F sensitivity of the organism. Experiments with permeabilized cells suggested that thiols themselves had no direct effect on the F sensitivity of enolase (a principal F target). Cells pretreated with DDT subsequently exhibited decreased F sensitivity when examined in the presence of saliva but not in the absence of saliva. Cells pretreated with whole salivary supernatant were found to be subsequently less sensitive to F in the absence of saliva during glucose uptake. Furthermore, in cases where cells were pretreated with saliva, subsequent additions of DDT were unnecessary to obtain maximal reduction in the F sensitivity of glucose uptake. It was concluded that the saliva-dependent reduction in F sensitivity of glucose uptake was not due to sequestration of available F by salivary constituents. The data suggest that a salivary component(s) interacts directly with the microorganism in some manner which results in reduced F sensitivity of the process under study. Possible mechanisms of saliva action are discussed.

Effect of bacterial aggregation on the adherence of oral streptococci to hydroxyapatite.

Several in vitro assay systems to measure the adherence of human dental plaque bacteria to solid surfaces such as teeth, glass, and hydroxyapatite have been published. In many studies a variety of macromolecular solutes have been used to study the adherence process. Often these solutes are able to aggregate the test bacterial and thus may alter the outcome of adherence experiments. In this study, the effects of the aggregation of Streptococcus sanguis on adherence to spheroidal hydroxyapatite is described. Adherence of preformed aggregates and of bacteria which were aggregating during the adherence reaction was examined. Bacteria were aggregated with whole saliva, concanavalin A, and wheat germ lectin. Further effects of the coaggregation of S. mitis and Actinomyces viscosus to saliva-coated spheroidal hydroxyapatite are presented. These studies suggest that formation of large aggregates resulted in a decrease in the numbers of organisms which adhered. In contrast, the formation of small aggregates actually increased the numbers of bacteria that adhered. All increases in adherent bacteria occurred at low concentrations of aggregating substance in which visible bacterial aggregation was not evident. The data indicate that adequate dose-response experiments must be performed to ensure that solutes used as probes to study adherence mechanisms do not affect the adherence simply as a result of aggregation of the test microorganisms.

Simple filter paper procedure for estimation of glucose uptake via group translocation by whole-cell suspensions of bacteria.

A simple and rapid filter paper technique is described for processing samples from glucose uptake studies with whole cells of gram-positive or gram-negative bacteria that transport glucose via group translocation. The procedure yields results equivalent to those obtained with a conventional membrane filtration method and requires no special filtration equipment or source of vacuum.