Retrieval of biofilms from the oral cavity.

With the use of the removable stents or bonded enamel piece models with or without a continuous bacterial layer, many in vitro or in vivo studies can be initiated. For example, studies on salivary pellicle formation, surface characteristics of biomaterials as they affect plaque development, antiplaque agents, the dynamics of adhesion of bacteria, interspecies adhesion of bacteria, the colonization of bacteria, the dynamics of bacterial growth in vivo, and the succession of growth in older supragingival plaques can be carried out.

Growth dynamics in a natural biofilm and its impact on oral disease management.

Measurements of the microbial growth dynamics in natural biofilm communities are almost non-existent. In a recent study, the biofilm formation on teeth was examined. A previously unknown active period of bacterial division occurred at a certain density of plaque bacteria on tooth enamel. The density-dependent cell-division phase of plaque formation contributed 90% of the biomass in the first 24 hrs of plaque formation. This suggested that growth was induced by the bacteria. In vitro assays were developed for rapid evaluation of the growth of surface-linked bacteria by the measurement of cellular components associated with growth on a per cell per time basis. Cell-free supernatants (termed START) of media in contact with bacteria were assayed for their effects on DNA synthesis and other cellular components associated with growth. START was found to increase the incorporation of [3H-methyl]-thymidine on a per cell per time basis, when compared with media not in contact with bacteria. Additional in vivo studies and in situ-based models of complex biofilms are needed if all of the mechanisms involved in the rapid accumulation of biofilm bacteria on teeth and other surfaces are to be understood.

Adherence, accumulation, and cell division of a natural adherent bacterial population.

Developing dental bacterial plaques formed in vivo on enamel surfaces were examined in specimens from 18 adult volunteers during the first day of plaque formation. An intraoral model placing enamel pieces onto teeth was used to study bacterial plaque populations developing naturally to various cell densities per square millimeter of surface area of the enamel (W. F. Liljemark, C. G. Bloomquist, C. L. Bandt, B. L. Philstrom, J. E. Hinrichs, and L. F. Wolff, Oral Microbiol. Immunol. 8:5-15, 1993). Radiolabeled nucleoside incorporation was used to measure DNA synthesis concurrent with the taking of standard viable cell counts of the plaque samples. Results showed that in vivo plaque formation began with the rapid adherence of bacteria until ca. 12 to 32% of the enamel's salivary pellicle was saturated (ca. 2.5 x 10(5) to 6.3 x 10(5) cells per mm2). The pioneer adherent species were predominantly those of the "sanguis streptococci." At the above-noted density, the bacteria present on the salivary pellicle incorporated low levels of radiolabeled nucleoside per viable cell. As bacterial numbers reached densities between 8.0 x 10(5) and 2.0 x 10(6) cells per mm2, there was a small increase in the incorporation of radiolabeled nucleosides per cell. At 2.5 x 10(6) to 4.0 x 10(6) cells per mm2 of enamel surface, there was a marked increase in the incorporation of radiolabeled nucleosides per cell which appeared to be cell-density dependent. The predominant species group in developing dental plaque films during density-dependent growth was the sanguis streptococci; however, most other species present showed similar patterns of increased DNA synthesis as the density noted above approached 2.5 x 10(6) to 4.0 x 10(6) cells per mm2.

Dental plaque development on defined streptococcal surfaces.

Coaggregations between bacterial species have been widely studied in vitro but not in the mouth. A new in vivo assay was used to measure the rate and composition of indigenous plaque formation onto bovine enamel chips covered with a continuous layer of bacteria. Chips were covered with Streptococcus oralis ATCC 10557, which coaggregated with many oral species, or Streptococcus gordonii S7, which did not coaggregate with these oral species, and placed in the mouth for 4 and 24 h. There were no differences in the number of most indigenous bacterial species isolated from the two streptococcal surfaces. However, the number of Actinomyces viscosus as a proportion of total Actinomyces spp. was significantly different on the two surfaces at 24 h. With the exception of Actinomyces naeslundii and A. viscosus removed from the S7 surface, all indigenous species increased significantly in number from 4 to 24 h, irrespective of the streptococcal surface. This study demonstrated that interbacterial coaggregation had only a limited effect on in vivo plaque development. Thus suggesting that environmental factors, growth or other adherence phenomena are dominant in in vivo plaque formation.

Comparison of the distribution of Actinomyces in dental plaque on inserted enamel and natural tooth surfaces in periodontal health and disease.

The distribution of Actinomyces naeslundii, Actinomyces viscosus and Actinomyces odontolyticus in healthy and diseased adult populations was studied in 3 different ways. First, supragingival plaque formation at 2 through 72 h was examined in 12 periodontally healthy adults using a removable pre-measured surface of enamel bonded to molars and premolars. Second, a cross-sectional examination of the composition of both supragingival and subgingival plaque of unknown age was conducted in 205 adults exhibiting periodontal health to moderate disease. Third, the effects of oral hygiene instruction and root planing on the subgingival microflora of a subset of 19 subjects with moderate periodontitis were examined. The evaluation of 12 adults revealed that the predominant species in early plaque formation (2, 4 and 8 h) was A. odontolyticus. A. viscosus and A. naeslundii were present in developing plaques in almost all subjects in 2-h plaque, but absent in half the subjects when 4-, 8- or 24-h plaque was examined. These two species significantly increased in numbers per mm2 enamel surface area in 48- and 72-h plaques. A. odontolyticus was not related to clinical signs of periodontal disease in 205 adults, and its subgingival proportions in plaque did not change following periodontal treatment of 19 individuals. A. naeslundii was found in significantly higher numbers in supragingival than subgingival plaques in the 205 adults examined. The mean proportion of A. naeslundii significantly decreased as the magnitude of probing depth and attachment loss increased. The proportions of A. naeslundii and A. viscosus were found to be significantly increased in subgingival plaques following periodontal treatment.

A follow-up case report of Actinobacillus actinomycetemcomitans in human periodontal disease.

The purpose of this investigation was to compare clinical and microbial parameters in a follow-up case report of adult subjects harboring Actinobacillus actinomycetemcomitans (Aa) with clinically matched subjects who did not have detectable Aa. 16 subjects with Aa and 16 subjects without Aa at the baseline examination were re-examined at an average of 46 months following collection of baseline data. Clinical measurements were recorded and subgingival plaque sampled and evaluated for microbial flora from each maxillary first molar. In 16 subjects with Aa at baseline, 4 sites in 3 subjects had detectable actinobacilli at the follow-up appointment. 26 sites in 13 individuals with Aa at baseline had a significantly increased gingival index at the follow-up visit (p less than or equal to 0.05), but there was no significant increase in probing depth or attachment loss. 32 sites in the 16 subjects without Aa at baseline still did not have detectable levels of this microorganism at the follow-up examination nor was there any significant difference between baseline and the follow-up appointment for the gingival index, probing depth and attachment level measurements. In subjects with Aa at baseline, 1 of 12 teeth without Aa and 5 of 20 teeth with Aa had been extracted prior to the follow-up visit. In this population group, having sites where Aa was detected, 6 of 9 teeth which had a probing depth greater than or equal to 5 mm were lost before the follow-up data collection appointment. In the control group, which did not have detectable Aa at baseline, 9 teeth with probing depths greater than or equal to 5 mm were not lost. These observations, although not proving, suggest in this population group, that deeper probing depths taken together with the presence of Aa may have placed an individual at greater risk of tooth loss.

Clustering of an outer membrane adhesin of Haemophilus parainfluenzae.

Haemophilus parainfluenzae synthesizes an outer membrane protein adhesin which mediates binding to oral streptococci, salivary pellicle, and neuraminidase-treated erythrocytes. An indirect gold labeling technique and immunoelectron microscopy verified the location of this outer membrane protein. Further, a clustering of gold particles was observed in irregular patches at the cell surface.

Comparison of plaque microflora between Chinese and Caucasian population groups.

This investigation was designed to compare the predominant plaque micro-organisms from a Chinese group of patients exhibiting periodontitis with an age-, sex- and periodontal disease-matched Caucasian group of patients. In addition to race, the 2 population groups differed with respect to diet and oral hygiene habits, or effectiveness at removing plaque. Clinical measurements were determined along with an evaluation for micro-organisms in supragingival and subgingival plaque. Although the Chinese and Caucasian population groups were similar with respect to composition of micro-organisms in subgingival plaque, notable differences were observed in supragingival plaque. The Chinese group had higher mean proportions of spirochetes, motile rods. Fusobacterium spp. and dark-pigmented Bacteroides species, while the Caucasian group had higher mean proportions of cocci, total Actinomyces spp., A. viscosus and total Streptococcus spp. in supragingival plaque. The microbial differences observed in supragingival plaque may be explained at least in part, if not totally, by the higher plaque index scores of the Chinese versus Caucasian population groups.

Effect of neuraminidase on the adherence to salivary pellicle of Streptococcus sanguis and Streptococcus mitis.

Neuraminidase-sensitive adherence to experimental salivary pellicles was studied using eight strains of Streptococcus sanguis and five strains of Streptococcus mitis. Approximately 60% of the strains of each species showed significantly lower adherence to neuraminidase-treated versus untreated saliva-coated hydroxyapatite. In addition, the adherence of several of these streptococcal strains to saliva-coated hydroxyapatite and neuraminidase-treated saliva-coated hydroxyapatite was inhibited using galactose and N-acetyl-D-galactosamine. Results from these studies suggested that several salivary receptors mediate adherence of these species.

Utilization of a continuous streptococcal surface to measure interbacterial adherence in vitro and in vivo.

Cell-to-cell interactions are essential for the formation of dental plaque. A continuous layer of Streptococcus sanguis SA-1 cells fixed to a solid surface has been used to evaluate interactions among this bacterium, Haemophilus parainfluenzae, and Streptococcus sobrinus. S. sanguis cells were attached to a Falcon 3001 tissue culture plates or bovine enamel chips, coated with a biological adhesive. Scanning electron microscopy of the chips showed the streptococci as a contiguous surface. Radiolabeled bacteria were used to measure a second-species interbacterial adherence to the streptococcal-coated culture plates. Strains of H. parainfluenzae known to coaggregate (strain HP-28) and not to coaggregate (strains HP-42 and HP-80), in suspension with S. sanguis strain SA-1, were studied for adherence. Ten-fold-higher numbers of coaggregating strain HP-28 adhered in vitro to the streptococcal layer than did the non-coaggregating strains. S. sobrinus strain 6715 did not show appreciable adherence to the S. sanguis surface. Saliva did not affect the adherence of coaggregating or non-coaggregating H. parainfluenzae strains to S. sanguis strain SA-1. Bovine enamel chips, coated with streptococci, mounted on modified orthodontic appliances and placed in the mouths of three volunteers, facilitated the measurement of interbacterial adherence in vivo of streptomycin-resistant strains of H. parainfluenzae (HP-28R or HP-42R). Suspensions of bacteria were placed into the mouth, distributed throughout, and expectorated. After 15 or 120 minutes, the appliance with the chips was removed, the chips sonified, and colony-forming units (CFU) of streptomycin-resistant haemophili determined per chip.(ABSTRACT TRUNCATED AT 250 WORDS)

Distribution of oral Haemophilus species in dental plaque from a large adult population.

The periodontal status of maxillary first molars in 284 young adults demonstrating near-health to early disease was evaluated, and supragingival and subgingival plaque samples were collected. Plaque samples were processed anaerobically, enumerated microscopically for bacterial morphotypes, and cultivated on various media to enumerate the microflora. Although haemophili were ubiquitous (recovered in 98.5 and 96.2% of the supragingival and subgingival plaque samples, respectively), 50% of the respective samples had proportions of less than or equal to 1.5% and less than or equal to 0.33% total Haemophilus spp. based on total cultivable microflora. To study the distribution of Haemophilus spp., 377 colonies were identified from modified chocolate agar (selective for oral haemophili) from 14 supragingival and corresponding subgingival samples from 14 subjects. The most prevalent species, Haemophilus parainfluenzae, was found in significantly higher proportions, based on total haemophili on modified chocolate agar, in supragingival and subgingival samples from teeth with shallower probing depths (less than or equal to 3.0 mm) versus deeper probing depths (greater than or equal to 3.0 mm). Additional statistically significant findings included Haemophilus segnis in higher proportions in supragingival samples from deeper sites, Haemophilus aphrophilus in higher proportions in subgingival samples from deeper sites, and Haemophilus paraphrophilus in higher proportions in subgingival samples from shallower sites. Scatter diagrams illustrating the bivariate distributions of proportions of haemophili with proportions of dark-pigmented Bacteroides spp., spirochetes, and streptococci demonstrated that high proportions of haemophili were never recovered from sites with high proportions of Bacteroides spp. or spirochetes. All levels of haemophili, however, were recovered from sites with all levels of streptococci. Two potential systems for interpreting haemophili data were hypothesized for predicting periodontal probing depths. There was highly significant agreement between the two systems. Small but statistically significant correlations were found between the gingival index, probing depth, and attachment level, and proportions of total Haemophilus species in the respective samples.

Isolation of a protein-containing cell surface component from Streptococcus sanguis which affects its adherence to saliva-coated hydroxyapatite.

The isolation and partial characterization of a protein-containing cell surface component from Streptococcus sanguis which blocks the adherence of this microbe to saliva-coated hydroxyapatite are described. Several methods of extraction were attempted. Sonication of whole cells and cell walls proved to be the most successful and yielded biologically active adherence-blocking components. The adherence-blocking ability of these components was effective in intraspecies blocking experiments. The extract obtained from cell walls of S. sanguis was examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and shown to contain one major and two to three minor bands when stained with Coomassie blue. The molecular weight of the major band was estimated to be 70,000 to 90,000. Gel filtration of the sonified cell wall extract on 10% agarose yielded two active adherence-blocking peaks, the void volume and a second peak.

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.

Aggregation and adherence of Streptococcus sanguis: role of human salivary immunoglobulin A.

Fourteen freshly isolated strains of Streptococcus sanguis were obtained from the dental plaque of five healthy adults. Whole saliva was collected concomitant with the plaque isolates from the five subjects, and a second whole saliva sample was collected 10 weeks later. All possible combinations of the first five saliva samples, the second five saliva samples, and 14 strains of bacteria were tested for aggregation. Of the 140 combinations examined, 108 of 140 (77%) of the strains aggregated with the first saliva samples and 95 of 140 (68%) aggregated with the second saliva samples. Overall, 72% of the strains aggregated with both the first and second saliva samples. Removal of immunoglobulin A (IgA) from these same salivas resulted in 38 of 108 (35%) of the aggregates decreasing in intensity with the first saliva samples and 27 of 95 (29%) of the aggregates decreasing in intensity with the second saliva samples. No aggregates increased in intensity with saliva samples when IgA had been removed. Removal of IgA from saliva also resulted in a mean decrease of 46% in adherence of S. sanguis to hydroxyapatite coated with the IgA-deficient saliva. Several strains of S. sanguis were shown to aggregate strongly with human salivary and colostral IgA. In addition, S. sanguis strain S7 showed a 31% stimulation of adherence to hydroxyapatite precoated with human salivary IgA over the uncoated controls. Stepwise removal of IgA from saliva resulted in a decrease in aggregation intensity from strong (4+) to weak (1+ to 2+). Similarly, the adherence of S. sanguis to hydroxyapatite coated with these saliva samples decreased linearly as the salivary IgA was depleted. Alternatively, the addition of a small quantity of salivary IgA (20 mug/ml) to progressively diluted saliva maintained a high level of adherence and strong aggregation until the saliva dilutions reached between 1:8 in the adherence experiments and 1:32 for the aggregations. These data indicate that salivary IgA may play an important role in the microbial ecology of human dental plaque formation.

Compounds which affect the adherence of Streptococcus sanguis and Streptococcus mutans to hydroxyapatite.

Several compounds were evaluated in an in vitro assay system for their ability to block the adherence of Streptococcus sanguis to saliva-coated hydroxyapatite and Streptococcus mutans to dextran-coated hydroxyapatitite. Fatty acids, ranging from C-12 to C-20, the enzyme amylase, chlorhexidine, human sera, and several serum proteins blocked S sanguis adherence to saliva-coated hydroxyapatite. Chlorhexidine blocked S mutans adherence to dextran-coated hydroxyapatite, but human sera and serum proteins did not. The effects of these compounds on the adherence of these organisms to hydroxyapatite may help in the development of specific plaque control methods for use in human populations.

Differential recovery of Streptococcus mutans from various mitis-salivarius agar preparations.

Recoveries of Streptococcus mutans from human dental plaque were lower when plated on mitis-salivarius agar obtained from Baltimore Biological Laboratories as compared with mitis-salivarius agar obtained from Difco Laboratories. However, no difference in recoveries of established laboratory strains of S. mutans was observed between these two agar preparations.