REGLAMENTO DE QUEJAS Y DENUNCIAS DEL INSTITUTO FEDERAL ELECTORAL

Enrichment cultures of subgingival plaque

Eight plaque samples were obtained from untreated pockets in as many periodontitis patients. The pockets were at least 8 mm deep and showed bleeding on probing. After removal of supragingival plaque, a sample of the pocket contents was taken with a dental scaler. The samples were immediately immersed in 5 ml of pre-reduced albumin medium. The medium contained per liter: 9 g of bovine albumin (Sigma Chemical Co., St. Louis, MO), 0.5 g proteose peptone (Difco Laboratories, Detroit, MI), and 0.5 g of trypticase peptone (BBL Microbiology Systems, Cockeysville, MD). The samples were transported into an anerobic isolator (Coy, Ann Arbor, MI) with an atmosphere of 91% N2, 5% CO2 and 4% H2 that was kept at 37°C. All further

procedures were carried out in the isolator. Plaque was suspended by vortexing for 1 min. In order to select for protein-degrading microbiota, enrichment cultures were started by inoculating 0.1 ml of the plaque suspension into 8-ml amounts of albumin medium of the above composition, and peptone medium. The peptone medium contained per liter 5 g proteose peptone (Difco Laboratories) and 5 g of trypticase peptone (BBL Microbiology Systems), but no albumin. After incubation for 7 days, 0.1-ml amounts of the enrichment cultures were transferred into the same volume of fresh medium. The proteolytic activities, pH values, optical densities at 550 nm and species composition of the cultures were determined after 3 enrichment steps.

Microbiological analysis

The samples from the mixed cultures were plated onto enriched-blood agar [18]. The plates were incubated for 7 days in the isolator. From a radial segment of the plate all colonies, but at least 25, were picked and subcultured for identification. Gram stain and cellular morphology were determined microscopically. Further identifications were done using API RapID 32A, API 32E, API 32STREP (all BioMerieux, Marcy l'Etoile, France).

Cell yields at varying concentrations of albumin

To estimate the effects of albumin on the cell production in the enrichment cultures, media with 0, 1.25, 2.5, 5, 10, and 20 g/l albumin were tested. Growth assays were performed in triplicate using 96-well microtiter plates (Flow Laboratories, McLean, VA). To each well, 200 µl of medium and 50 µl of a 24-h preculture of subgingival plaque were added. After growth for 3 days, absorbance values at 540 nm (A540 nm) were

recorded using a Titertek Multiskan MC (Flow Laboratories). Residual albumin in the cultures was examined by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE).

Proteolytic activity

For determination of proteolytic activities, the cultures were centrifuged at 20,000 × g for 5 min, washed and resuspended in saline to a A550 nm value of 1.0. Using well

plates, the reaction mixture consisted of 50 µl of cell suspension, 25 µl of 0.4% resorufin-labelled casein (Boehringer, Mannheim) in water and 25 µl of 0.2 mol/L Tris-HCl buffer pH 7.8. After incubation for 4 h, the reaction was stopped by addition of 250 µl of 5% trichloroacetic acid, and the reaction mixture was centrifuged. Finally, 200 µl of the supernatant was added to 0.3 ml of 0.5 mol/l Tris-HCl pH 8.8, and absorbancies were read at 574 nm. Proteolytic activity was expressed as unit of A574 nm per unit of cell density (unit A550 nm).

The ability of individual strains to degrade albumin was measured after 7 days of incubation at 37°C in medium containing 1 g/l albumin.

Albumin assay

To determine the proteolytic activities of strains, albumin was assayed in the incubation mixtures using SDS-PAGE [14, 16]. Samples of the bacterial cultures were diluted in water, and boiled for 5 min with an equal volume of sample application buffer containing 1% SDS, 2 moles/l urea and 50 mmol/l dithiothreitol, in addition to the normal constituents. A nominal amount of approximately 4 g of albumin was loaded on the gel. The samples were electrophoresed with 3% stack gel at 40 mA.

Gels were stained with coomassie blue, and destained in 7% acetic acid and 5% methanol. Densitometry was performed directly on the wet gels by a laser densitometer (Pharmacia-LKB GSXL, Bromma, Sweden). The amount of degraded albumin was determined by comparison with the internal albumin standard on each gel.

Residual albumin in cultures was assayed by capillary electrophoresis (P/ACE System 2210, Beckman, Fullerton, CA) on a 27-cm coated glass column with an internal diameter of 50 µm (Applied Biosystems, Foster, CA). The system was provided with a an ultraviolet detector (214 nm). Separations were carried out at 10 kV in 0.1 mol/l phosphate buffer pH 2.5.

RESULTS

Enrichment cultures

The mean cell yield (A550 nm) of eight enrichment cultures originating from subgingival

plaque samples in medium with 10 g/l albumin was 0.88 (SD 0.40; Table 1). Assuming a mean molecular weight of 100 for amino acids, the cell yield is 0.0088 A550 nm

units/mol amino acid. The pH values of the enrichments in albumin ranged from pH 7.3 to pH 7.9. No residual albumin was found in the cultures, indicating that albumin was limiting the growth of the cultures. Some samples were also enriched in medium with 10 g/l peptone (Difco Labs) instead of albumin. The cell yields in the hydrolyzed protein, peptone, were found to be equally high as in albumin medium ( Table 1).

Table 1. Cell yield and proteinase activities of mixed culures of periodontal bacteria. Means and standard deviations in brackets

Table 2. Isolates from enrichment cultures of subgingival plaque in albumin medium

and their proteinase activities

Microorganisms in enrichment cultures and proteolytic activities

Microorganisms isolated from eight enrichments of individual subgingival plaque samples are listed in Table 2. Peptostreptococcus micros, Prevotella buccae,

Fusobacterium nucleatum, Streptococcus gordonii and Streptococcus anginosus

frequently occurred in the eight enrichment cultures. The microbial composition of a representative enrichment culture, enrichment culture no. 3 (see Fig. 1, at 10 g/l albumin), was as follows: P. micros, 9%; P. buccae, 60%; F. nucleatum, 10 %;

Actinomyces meyeri, 3%; S. gordonii, 9%; and S. anginosus, 9%. Proteinase activities,

measured with resorufin casein and by degradation of albumin, was found in some species but not in others.

The proteolytic activities of the cellular and supernatant fractions of mixed cultures are shown in Table 1. The activities in the albumin cultures were noticeably higher than those in the peptone cultures. In both media, most of the proteolytic activity that occurred was cell associated.

Albumin as a growth-limiting substrate

A linear relationship was found between the cell mass (units A550 nm) in plaque

example is given in Fig. 1. No residual albumin was found in the cultures, up to the highest concentration (20 g/l) tested. Individual isolates from the same plaque enrichment culture were grown in the albumin medium. Fig. 1 indicated that the pure cultures of the isolates grew poorly, in contrast to their mixture.

Figure. 1. Cell mass of enrichment culture (E. culture number 3) and cell masses of pure cultures of its isolates in

media with increasing concentration of albumin. The microbial composition of the enrichment culture number 3 is detailed in the text.

DISCUSSION

Proteins from crevicular fluid seem to be the major energy source for growth of the periodontal microflora. This focuses the attention on the role of proteolytic enzymes in the acquisition of substrate. Because of its predominance in crevicular fluid, albumin was selected as substrate for enrichments of subgingival plaque. Using a single protein instead of serum [18], it was anticipated that relatively simple mixed cultures would be obtained.

This appeared to be the case, and albumin supported growth of 5–8 membered mixed cultures originating from subgingival plaque samples. The complete utilization of albumin in the cultures suggested that albumin acted as growth-limiting substrate. This was confirmed by the linear relationship between cell yield and albumin concentration. Assuming a mean molecular weight of amino acid of 100, the cell yield of the mixed cultures was approximately 0.09 A550 nm units/mol amino acid. Growth of

oral streptococci and actinomyces species on hexose gives yields of approximately 0.1 A550 nm units/mol hexose [8, 21]. The higher efficiency of hexose than amino acid

as energy substrate for mixed growth of oral bacteria corraborates findings in other ecosystems [3, 10] . The reasons are that only some amino acids can be fermented, and that the molar ATP yield of amino acid fermentation is generally lower than that of hexose.

That most of the proteinase activity occurred cell-associated is a common observation for periodontal microflora [6, 11]. Most of the proteolytic activity in the rumen and the large intestine also resides in the bacterial fraction [15, 22]. The stimulation of proteinase activities by albumin in the medium suggested that proteinase was induced.

All eight albumin enrichment cultures had some crucial characteristics in common. Each contained one or more proteinase-producing bacteria, including Prevotella spp. and A. meyeri. Peptostreptococcus and Fusobacterium occurred in most cultures. Their presence is thought to be due to the availability of polypeptides generated by the proteinase-producing strains. The degradation of albumin by P. micros, as found here, seems in contrast with other observations indicating the absence of proteinase activity [12] in this species. Instead, albumin breakdown by P. micros may have resulted from endopeptidase activity and the long incubation (1 week) in the test that was used here.

Since the carbohydrate content of the albumin medium was low, the frequent occurrence of typical carbohydrate fermenters as S. anginosus and S. gordonii is interesting. In fact, these streptococcal species are versatile and, in addition to the fermentation of different sugars, they have the ability to ferment arginine [7, 20]. This may explain their common appearance in various sites in the mouth. Other species, such as Gemella, do not seem to ferment amino acid, and their presence may be due to the small amount of carbohydrate in the medium.

The mixed culture grew much better than would be expected from growth of the individual strains. This strongly suggested that microorganisms synergistically degrade albumin by overlapping proteolytic (proteinase and peptidase) activities.

Overall, the albumin enrichment cultures have some basal characteristics in common with natural subgingival plaque. Disease-associated microorganisms such as Prevotella, Porphyromonas, Peptostreptococcus, Fusobacterium and Treponema use amino acids as energy source and rely on proteolytic activities to fulfil their amino acid requirements. This would make it interesting to estimate the effect of proteinase inhibitors on the subgingival microflora. The albumin enrichment cultures may serve as a relatively simple in vitro model to evaluate the effects of proteinase inhibitors.

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