Characterisation of Bacteroides from sheep periodontal disease by SDS-PAGE of outer membrane proteins.

Isolates of Bacteroides species obtained from a longitudinal study of developing periodontal disease in sheep were analysed by SDS-PAGE. Protein profiles of Sarkosyl-insoluble outer membrane extracts were compared within groups of isolates which had already been defined by conventional biochemical techniques. Heterogeneity was exhibited within most groups. Isolates of B. gingivalis and B. asaccharolyticus shown to be similar to human isolates by conventional biochemical tests, gave different protein profiles from the respective type cultures. The sheep B. gingivalis-like isolates were however homogeneous, while the B. asaccharolyticus-like organisms could be divided into 3 subgroups. SDS-PAGE appears to be a useful tool for the examination of bacterial flora and recognition of subgroups of subspecies.

A longitudinal study of the cultivable subgingival anaerobic bacteria isolated from sheep during the development of broken mouth periodontitis.

In a longitudinal bacteriological study of the cultivable subgingival anaerobic flora isolated from developing broken mouth periodontitis in sheep, samples were taken from five sheep on each of three farms on seven occasions over a period of 2.5 years. Ten different bacterial genera were isolated regularly but with fluctuating frequencies. Bacteroides and Fusobacterium organisms accounted for nearly 70% of the isolates. The Bacteroides and Fusobacterium isolates studied in detail from one farm were identified to species level. The fusobacteria comprised F. nucleatum-like organisms (68.6%). F. necrophorum (29.6%) and F. naviforme (1.8%). The Bacteroides spp. were divided into 11 main groups and included black-pigmented species similar to B. asaccharolyticus and B. gingivalis. On the farm studied in detail, the sheep could be allocated to two groups according to progression of periodontal disease. Most of the B. gingivalis-like isolates were from sheep with actively progressing disease, indicating that this organism may play a role in periodontal destruction in sheep.

Preliminary study of the anaerobic bacteria isolated from subgingival plaque from sheep.

A preliminary study was made to determine the genera of cultivable anaerobic bacteria which could be isolated from subgingival plaque of sheep. Samples were taken from 10 sheep on farms with a known record of broken mouth periodontitis. For assessment of the sampling technique, samples were also taken from freshly exposed tooth roots in killed sheep. The bacteria isolated on several selective and non-selective media were identified to genus level by a combination of Gram reaction, colony morphology and gas chromatographic analysis of volatile and non-volatile fatty acid metabolic end products. At least 10 different genera were isolated and these findings are discussed in relation to the bacteriology of human periodontitis and recent studies of sheep broken mouth periodontitis.

Longitudinal study of untreated periodontitis (II). Microbiological findings.

11 volunteer subjects with advanced chronic periodontitis participated in a 1-year longitudinal clinical and microbiological study. Subgingival plaque was collected at each of 7 visits from 148 pre-selected sites in the left jaw quadrants (test sites) and on the first and last visits, only from 117 sites in the left jaw quadrants (control sites). All sites were examined clinically at each of the 7 visits, and the microbiological markers investigated were the % spirochaetes and % black pigmented Bacteroides species in subgingival plaque. At the completion of the study, the sequential changes in probing attachment level at each site were subjected to regression analysis to determine the direction and extent of attachment change. Possible correlations between attachment change and % spirochaetes or % black-pigmented bacteroides were investigated using both individual sites and individual subjects. No significant differences were observed in either of the microbial variables between test and control sites. Possible correlations between the microbiological markers and attachment changes were investigated at baseline, at the 12-month visit and using the microbial data accumulated over all 7 visits. Significant differences were observed only at the 12-month visit when the % spirochaetes of both test and control sites were significantly lower in subjects showing the greatest improvement in attachment level. Overall, these results indicate that quantification of either spirochaetes or black-pigmented Bacteroides species cannot be used reliably to identify or predict disease-active sites.

A new method for assessing the % of spirochaetes in subgingival plaque.

A new method for counting the numbers of spirochaetes in subgingival plaque is described. The technique involves the combined use of a negative stained preparation, dark field microscopy, and an image analysing system. Advantages of the method include: smears need not be made until 2 days after sampling, and can be examined microscopically for up to 1 year after preparation; reproducibility of counts are more than 90% and the data can be computerised. The new method should prove useful in the routine assessment of subgingival plaque from patients with various forms of periodontitis, and in epidemiological surveys.

Effect of saliva and serum on the adherence of Candida species to chlorhexidine-treated denture acrylic.

The effect of saliva and serum on the adherence of five strains of Candida albicans and one each of C. tropicalis and C. glabrata to chlorhexidine-pretreated acrylic was measured in vitro. A four-fold dilution of saliva or serum significantly inactivated the fungicidal effect of chlorhexidine gluconate. Pretreatment of the acrylic with unstimulated mixed saliva for 30 min led to a reduced adherence for all the Candida strains tested, whilst a similar pretreatment with serum slightly increased adhesion. Moreover treatment of saliva- or serum-coated acrylic with chlorhexidine gluconate 2% reduced adherence by between 19% and 86%. The inhibition of yeast adherence by chlorhexidine persisted for up to 19 days after the exposure of the acrylic strips to the disinfectant.

A comparison of the effects of chlorhexidine gluconate, amphotericin B and nystatin on the adherence of Candida species to denture acrylic.

Pretreatment of denture acrylic with nystatin, amphotericin B and chlorhexidine gluconate significantly reduced the subsequent adherence of Candida species to acrylic, although chlorhexidine and nystatin were more effective than amphotericin B. The duration of chlorhexidine-mediated inhibition of adherence (up to 19 days) was much longer than that of amphotericin B (24 h) or nystatin (48 h). Exposure of stationary-phase cells of Candida species for a short period to sublethal concentrations of chlorhexidine, or growth of Candida species with sublethal concentrations of chlorhexidine for 24 h resulted in a reduction in the ability of the yeasts to adhere to denture acrylic. The organisms grown in the presence of chlorhexidine were more susceptible to spheroplasting with Zymolyase 20T, indicating that chlorhexidine affects the cell surface composition of Candida species.

Effect of chlorhexidine gluconate on the adherence of Candida species to denture acrylic.

The effect of pretreatment of denture acrylic with chlorhexidine gluconate on the subsequent adherence of Candida albicans GDH 2346 was measured in vitro. Adherence was significantly reduced by pretreatment with chlorhexidine; maximal inhibition was achieved by incubation at room temperature for 30 min in 2% chlorhexidine. Inhibition of adherence was greatest when the organisms were grown in conditions that enhanced adherence the most, i.e., growth to stationary phase in high concentrations of galactose and sucrose. Yeasts grown in high concentrations of galactose, which were the most adherent to acrylic, were also the most sensitive to the fungicidal action of chlorhexidine gluconate, whereas those grown in a low concentration of glucose were the least adherent and also the most resistant. Adherence to acrylic of seven strains of C. albicans isolated from active infections (I strains) and grown in medium containing 500 mM sucrose was significantly higher than that of four strains obtained from asymptomatic carriers (C strains). A spectrum of adherence values was obtained when various yeasts other than C. albicans were tested.

Extracellular polymer of Candida albicans: isolation, analysis and role in adhesion.

Extracellular polymeric material (EP) was isolated from culture supernatants of Candida albicans grown on carbon sources (50 mM-glucose, 500 mM-sucrose or 500 mM-galactose) known to promote yeast adhesion to different extents. Galactose-grown yeasts, which are the most adherent, produced more EP than sucrose-grown organisms, particularly after incubation for 5 d, while glucose-grown yeasts (the least adherent) gave the lowest yield. EP produced on all three carbon sources was of similar composition and contained carbohydrate (65 to 82%; mannose with some glucose), protein (7%), phosphorus (0.5%) and glucosamine (1.5%). Serological studies indicated that these EP preparations were immunologically identical but that galactose-grown yeasts had more antigenic determinants than sucrose-grown organisms while glucose-grown yeasts had the fewest determinants. Antigenic differences were apparent between EP preparations of some strains of C. albicans. Pretreatment of acrylic strips with EP to form a polymeric coating promoted yeast adhesion to the acrylic surface, but similar pretreatment of buccal epithelial cells with EP inhibited subsequent yeast adhesion. These results indicate that EP originates from the cell surface of C. albicans and that it contains the surface component(s), probably mannoprotein in nature, responsible for yeast adhesion.

Relationship between cell surface composition, adherence, and virulence of Candida albicans.

A comparison was made of the adherence to acrylic and to human buccal epithelial cells of seven strains of Candida albicans isolated from active infections (I strains) and two strains obtained from asymptomatic carriers (C strains). After growth in defined medium containing a relatively low concentration (50 mM) of glucose as the carbon source, the adherence of I and C strains to either surface was similar and all strains were sensitive to spheroplast formation with Zymolyase 5000. Growth in medium containing a high concentration (500 mM) of sucrose or galactose enhanced the adherence of I strains up to 5- and 11-fold, respectively, and there were corresponding increases in resistance to spheroplast formation. Sucrose- or galactose-grown C strains showed only small increases in adherence and remained relatively sensitive to spheroplast formation. When inoculated intravenously into mice, I strains grown in 500 mM sucrose were up to five times more virulent than organisms grown in 50 mM glucose, while I strains grown in 500 mM galactose showed a 5- to 24-fold increase in virulence. Fifty percent lethal doses obtained for C strains were similar after growth on all three carbon sources. We conclude that I strains are able to modify their surface composition in response to high concentrations of certain sugars in the growth environment. Such modification can enhance both their ability to adhere to surfaces and their virulence. C strains lack this capability, or possess it to a lower degree, and may therefore have a lower pathogenic potential.

Relationship between cell surface composition of Candida albicans and adherence to acrylic after growth on different carbon sources.

The adherence of Candida albicans to acrylic was measured in vitro after growth of the yeast to stationary phase in defined medium containing glucose, sucrose, galactose, fructose, or maltose as the carbon source. In each case, yeast adherence was proportional to the concentration of sugar in the growth medium, but equimolar concentrations of different sugars promoted adherence to different extents. In vitro adherence was further increased by the addition of divalent cations to assay mixtures but was inhibited when saliva-treated acrylic strips were used or when yeasts were suspended in mixed saliva during the assay. The rate of spheroplast formation of yeasts grown in media containing a 500 mM concentration of the different sugars correlated well with the relative adherence of the cells to acrylic. Galactose-grown yeasts were most resistant to spheroplast formation with Zymolyase-5000 and most adherent to acrylic, whereas fructose-grown organisms were least resistant to spheroplast formation and least adherent to acrylic. These results indicate that when grown to stationary phase in media containing high concentrations of certain sugars, C. albicans undergoes a change in cell surface composition which facilitates its adherence to acrylic surfaces. Electron microscopy of yeasts harvested from such media revealed the presence of an additional surface layer which may be responsible for this enhanced adherence.