M.H. de Jong

The Growth of Oral Bacteria on Saliva

The present experiments were aimed at studying the degradation of salivary glycoproteins by the oral microflora. To this end, S. sanguis I strain Ny476 and S. sanguis II (S. mitior) strain Ny581 were grown continuously in human-whole saliva. Under these conditions, the strains produced a variety of cell-associated hydrolytic activities, including glycosidases, exo- and endopeptidases, and esterases. S. sanguis II generally exhibited higher levels of enzyme activity than did S. sanguis I, in particular of neuraminidase that was produced only by S. sanguis II. In accordance, S. sanguis II had a higher cell yield and consumed a higher proportion of the sugars and sialic acid in the glycoproteins than did S. sanguis I. Interestinglv, S. sanguis I, which is devoid of neuraminidase, is known to have a lectin with specificity for sialic acid, whereas S. sanguis II has affinity for galactose residues in the glycoproteins. We propose that specific binding of glycoproteins by oral bacteria constitutes a mechanism to collect nutrients in the vicinity of the cell. The special ability of S. sanguis II to utilize saliva for growth was further exemplified by its selection in batch-wise enrichments of dental plaque on saliva. The microflora in these enrichment cultures always consisted of Peptostreptococcus micros, S. sanguis II, and Fusobacterium nucleatum as the dominant organisms. Further, S. mitis and Gemella hæmolysans were generally found to be present. The enrichment cultures produced a wide variety of mainly cell-bound hydrolytic enzymes. This resulted in almost complete breakdown of salivary glycoproteins in the culture.The present experiments were aimed at studying the degradation of salivary glycoproteins by the oral microflora. To this end, S. sanguis I strain Ny476 and S. sanguis II (S. mitior) strain Ny581 were grown continuously in human-whole saliva. Under these conditions, the strains produced a variety of cell-associated hydrolytic activities, including glycosidases, exo- and endopeptidases, and esterases. S. sanguis II generally exhibited higher levels of enzyme activity than did S. sanguis I, in particular of neuraminidase that was produced only by S. sanguis II. In accordance, S. sanguis II had a higher cell yield and consumed a higher proportion of the sugars and sialic acid in the glycoproteins than did S. sanguis I. Interestinglv, S. sanguis I, which is devoid of neuraminidase, is known to have a lectin with specificity for sialic acid, whereas S. sanguis II has affinity for galactose residues in the glycoproteins. We propose that specific binding of glycoproteins by oral bacteria constitutes a mechanism t...

Enrichment of subgingival microflora on human serum leading to accumulation of Bacteroides species, Peptostreptococci and Fusobacteria

This study was undertaken to identify ecological factors that favour opportunistic pathogenic species in the subgingival microflora. In a first approach, human serum as a substitute for gingival exudate, was used for batch-wise enrichment of subgingival plaque. The microflora resulting after 5–6 enrichment steps consisted of black-pigmented and non-black-pigmented Bacteroides species, Peptostreptococcus micros and Fusobacterium nucleatum as the main organisms. It is noted that the same group of species was found to be enriched independent upon the origin of the subgingival plaque sample. It was suggested that these organisms are favoured by the increased flow of gingival exudate during inflammation.The consortium of organisms was capable of selective degradation of serum (glyco-)proteins. Four different types of degradation occurred. After a prolonged period of growth complete degradation of immunoglobulins, haptoglobin, transferrin and complement C3c was observed. Partial degradation of immunoglobulins, haptoglobin, transferrin, albumin, alpha1-antitrypsin and complement C3c and C4 was generally observed after 48 h of growth. Besides, immunoglobulin protease activity yielding Fc and Fab fragments was found. The consortium was also capable of consuming carbohydrate side-chains as indicated by an altered electrophoretic mobility of the serum glycoproteins.

Clinical Science Effects of Highly Concentrated Stannous Fluoride and Chlorhexidine Regimes on Human Dental Plaque Flora

The aim of this study was to determine the effect of an intensive antimicrobial treatment on the number of Streptococcus mutans, Streptococcus sanguis, Actinomyces viscosus/Actinomyces naeslundii, and the total Colony-forming Units (CFU) in plaque. The dentition of human volunteers was treated in a dental office with either chlorhexidine (5%) or stannous fluoride (8%). Following the office treatment with chlorhexidine, selected volunteers rinsed daily at home for seven or 49 days with chlorhexidine solution (0.2%), while another group flossed daily at home for seven days with dental floss impregnated with chlorhexidine. On days one, seven, 21, 35, and 49 after the local applications, we took saliva samples and plaque samples from fissures, smooth surfaces, and approximal areas. Chlorhexidine and stannous fluoride suppressed S. mutans and the Actinomyces species on all surfaces and in saliva. S. mutans on tooth surfaces was suppressed for approximately seven days and returned to the baseline level at day 21. A. viscosus/naeslundii was suppressed for more than seven days on the teeth. S. sanguis and the total CFU returned to the baseline level within seven days on all surfaces and in saliva. Rinsing or flossing with chlorhexidine suppressed S. mutans during the period of time that these supplements were used. Brushing for seven days with chlorhexidine gel (1%) without a preceding intensive chlorhexidine treatment had virtually no effect on S. mutans in approximal areas and in saliva, but suppressed S. mutans in fissures and on smooth surfaces.

Growth of Micro-organisms from Supragingival Dental Plaque on Saliva Agar

The role of saliva in supporting the growth of dental plaque has scarcely been investigated. We have studied the growth and recovery of micro-organisms from dental plaque samples on saliva-agar plates, prepared from filter-sterilized wax-stimulated whole saliva. Under optimal conditions, the mean recovery of plaque samples on saliva agar was about 50% (range, 22-77) of the recovery on blood agar. Addition of 2.5 mmol/L dithiothreitol (DTT) strongly facilitated filter-sterilization of saliva, but DTT concentrations higher than 1 mmol/L reduced the recovery of plaque micro-organisms on saliva agar.Catalase (100 U/ml) offered protection against the inhibitory effect of DTT. Addition of glucose (1-5 mmol/L) increased only slightly the recovery of plaque micro-organisms on saliva agar. When supragingival plaque was plated on saliva- and blood-agar plates, the composition of the microflora isolated from saliva agar strongly resembled that isolated from blood agar. The predominant species — i.e., Streptococcus a...The role of saliva in supporting the growth of dental plaque has scarcely been investigated. We have studied the growth and recovery of micro-organisms from dental plaque samples on saliva-agar plates, prepared from filter-sterilized wax-stimulated whole saliva. Under optimal conditions, the mean recovery of plaque samples on saliva agar was about 50% (range, 22-77) of the recovery on blood agar. Addition of 2.5 mmol/L dithiothreitol (DTT) strongly facilitated filter-sterilization of saliva, but DTT concentrations higher than 1 mmol/L reduced the recovery of plaque micro-organisms on saliva agar. Catalase (100 U/ml) offered protection against the inhibitory effect of DTT. Addition of glucose (1-5 mmol/L) increased only slightly the recovery of plaque micro-organisms on saliva agar. When supragingival plaque was plated on saliva- and blood-agar plates, the composition of the microflora isolated from saliva agar strongly resembled that isolated from blood agar. The predominant species — i.e., Streptococcus and Actinomyces — all grew on saliva agar though usually in numbers somewhat lower than those on blood agar. In addition, most species normally found only in low proportions in supragingival dental plaque were also found to grow on saliva agar. Collectively, the results lead to the conclusion that the supragingival microflora can utilize saliva as a complete source of nutrients.

A Conceptual Model for the Co-existence of Streptococcus Spp. and Actinomyces Spp. in Dental Plaque

One of the most important questions in ecology is how to explain the co-existence of the variety of physiologically related organisms in the same habitat. A model is presented for the co-existence of Streptococcus species and Actinomyces species in dental plaque. The hypothesis is that these organisms co-exist because they simultaneously utilize several carbon and energy substrates. The hypothesis follows from the observation that the growth yield of oral streptococci and actinomyces in saliva is limited by carbohydrate. Preliminary experiments were undertaken to test the hypothesis using mixed chemostat cultures and gnotobiotic rats. Competition between S. mutans K1R and A. viscosus Ut2 in mixed chemostat cultures on glucose and asparagine was hampered by the early appearance of high-glucose-affinity variants of A. viscosus. From the physiological characteristics of S. sanguis and S. milleri, it might be predicted that simultaneous utilization of carbohydrate and arginine would enable these organisms to co-exist with S. mutans in an ecosystem. To test this mechanism under natural conditions, germ-free rats were inoculated with a combination of S. mutans K1R and S. sanguis P4A7 or the combination S. mutans K1R and S. milleri B448. The rats were fed on three different diets: (1) 58% cornstarch ; (2) 48% cornstarch and 10% sucrose; and (3) 53% cornstarch and 5% arginine. The results of this experiment demonstrated that dietary arginine caused a significant decrease of the ratios K1R/P4A7 and K1R/B448 in dental plaque. Thus, arginine stimulated S. sanguis and S. milleri, whereas sucrose promoted S. mutans. It can be concluded that bacterial substrate is a major ecological factor for dental plaque. This factor can potentially be studied in mixed-chemostat cultures and in gnotobiotic rats.

Effects of Dietary Carbohydrates on the Numbers of Streptococcus mutans and Actinomyces viscosus in Dental Plaque of Mono-infected Gnotobiotic Rats

Dietary sucrose and glucose are known to promote the accumulation of, respectively, Streptococcus mutans and Actinomyces viscosus in complex dental plaque microflora. In this study, we have investigated the effects of these carbohydrates on the numbers of S. mutans and A. viscosus colonizing the teeth of mono-infected gnotobiotic rats. Sucrose at a dietary concentration of between 0.25 and 0.5 percent (corresponding with 7-15 mM in the drinking water) caused an increase in the numbers of S. mutans. This result strongly suggested that the increase was due to extracellular polysaccharide (EPS) synthesis by glucosyltransferase, whose Km value (1.0-7.0 mM) is in the same concentration range. The numbers of A. viscosus increased three-fold when 10% glucose was added to the basal diet. It seems plausible that this was due to extracellular heteropolysaccharide produced by A. viscosus in the presence of excess glucose. Although in most microbial ecosystems provision of additional substrates leads to an increase in biomass, glucose had only a small effect on the numbers of S. mutans. Mechanical dislodging forces could be limiting the accumulation of dental plaque. EPS synthesis from dietary carbohydrates seems to enable the population to withstand mechanical dislodging at least partially by providing a matrix wherein cells are entrapped or to which the cells are firmly attached.

Colonization of the teeth of rats by human and rodent oral strains of the bacterium Actinomyces viscosus

The mouths of rats were infected with human and rodent strains of A. viscosus and streptomycin-rifamycin-resistant (SR) mutants of these strains. The establishment, adherence to the teeth and cell dose required for infection were determined. Human and rodent strains established equally well on the teeth and did not differ in their initial adherence to teeth. The cell dose required for infection was much lower for rodent than for human strains (less than 10(5) compared to 10(7) - 10(8) c.f.u.). The SR mutants established as well as their parents except for one SR strain, that did not establish at all. Another SR mutant appeared to have lost its SR resistance after passage through the animal. The results stress the need of in-vivo testing of antibiotic resistant mutants to be used in ecological studies.

Effects of chlorhexidine, iodine, and 5,7-dichloro-8-hydroxyquinoline on the bacterial composition of rat plaque in vivo.

The effects of short-term high-dose therapies with chlorhexidine, iodine, or 5,7-dichloro-8-hydroxyquinoline (DCHQ) on the microbial composition of dental plaque in conventional rats were compared. Th

The pattern of experimental colonization of a human and a rodent strain of the bacterium Actinomyces viscosus on the dentition of the rat

Samples were taken from mesial, buccal, lingual and approximal sites and from fissures. Initially, most A. viscosus were recovered from the retention sites. With the exception of lingual sites during the period of exponential growth, the apparent doubling times calculated for A. viscosus Nyl SR remained within narrow limits for all locations. After cessation of exponentional growth, both strains had colonized all surfaces. However, the rodent strain A. viscosus Nyl SR had formed 30-800 times larger populations on the smooth surfaces than the human strain A. viscosus Ut2 . On the retention sites, the populations of both strains were not significantly different.

The Effect of Chlorhexidine on the Colonization of the Human and Rat Dentition by Actinomyces viscosus

The dentition of humans and rats was treated with a short-term, high-dose application of chlorhexidine. This strongly suppressed the indigenous microflora on the teeth. At different time intervals aft