Plaque Formers Research Articles

Clonal Relationship within Two Oral Actinomyces Populations Collected from Plaque of Periodontitis Patients

Actinomyces naeslundii and A. oris are dental plaque formers involved in the pathogenesis of periodontitis. The aim of the study was to investigate the clonal relationship within two oral Actinomyces populations collected from plaque of patients with chronic periodontitis. The 223 clinical strains of A. naeslundii and A. oris were isolated from biofilm samples collected supra and subgingivally from teeth with shallow (probing depth (PD) = 3-4 mm), deep (PD = 5-6 mm) and very deep (PD ≥7 mm) pockets from 20 chronic periodontitis patients. All strains were submitted to repetitive sequence-based PCR typing using DiversiLab (BioMerieux,Marcy l´Étoile, France). Seven patients harboured only unrelated (<95% similarity) multiple isolates, while 13 harboured both similar (>95% similarity) and unrelated isolates at different sites. Identical (>98% similarity) strains were found to be present in the subgingival shallow depths more often than in the other subgingival depths. The number of clones in individual patients varied from 2 to 17 different rep-PCR genotypes. The clonal relationship within the oral populations of A. naeslundii and A. oris in an individual was unpredictable, ranging from the presence of multiple genotypes with no clonal similarity to only two different clones supra or subgingivally at different sites.

Two Randomized Trials Demonstrate Lactic Acid Supplementation in Pet Food Inhibits Dental Plaque, Calculus, and Tooth Stain in Cats.

Background:Dental concerns are some of the most common health problems affecting companion animals. A variety of foods, treats, and chews comprising different mechanical and chemical technologies have been investigated as a means of promoting oral health. Here, we investigate the chemical technology, lactic acid added to a commercially available food, for its ability to inhibit dental plaque, calculus, and tooth stain accumulation in cats.Methods:Two separate feeding trials assessed the utility of a nutritionally complete feline maintenance food supplemented with lactic acid to reduce oral substrate accumulation (dental plaque, calculus, and tooth stain) in cats. After a calibration study identified high and low dental plaque formers, 45 cats were randomized to 1 of 2 test groups (food with 1.2% lactic acid supplementation) or control (food without lactic acid supplementation) groups, stratified based on their calibration scores. Data were collected on a monthly basis for 3 months. The second study randomly assigned 24 cats to either the test or control groups for 1 year, with data collected at the 6- and 12-month time points.Results:In the 3-month study, reductions in dental plaque, calculus, and tooth stain accumulations were observed at the 2-month assessment in both test groups compared with control (P < .05 for test group 2). The 1-year study showed that these reductions in oral substrate accumulation persisted through the 6- and 12-month time points (P < .05).Conclusions:Taken together, these studies demonstrate that lactic acid supplemented at 1.2% in a feline maintenance food significantly inhibits oral substrate accumulation.

Prevalence of Actinomyces spp. in patients with chronic periodontitis

This study investigated the prevalence of Actinomyces spp. in shallow, deep and very deep pockets of patients with chronic periodontitis compared to healthy controls and correlated the results with clinical status. Twenty patients with chronic periodontitis and 15 healthy subjects were enrolled in this study. Clinical indices were recorded in a six-point measurement per tooth. From each patient samples of supra and subgingival plaque were taken separately from teeth with shallow, deep and very deep pockets. Samples of supragingival plaque and sulcular microflora were collected from the healthy subjects. All the samples were cultivated on different media at 37̊C in an anaerobic atmosphere for 7 days. All the suspect colonies were identified using a rapid ID 32 A system (bioMèrieux) and MALDI-TOF-MS analysis using an Autoflex II Instrument (Bruker Daltonics) together with in house developed identification software and a reference spectra database. A total of 977 strains were identified as Actinomyces. Actinomyces naeslundii/oris/johnsonii (430 isolates) was the most prevalent species and was found in all patients and in almost all of the healthy subjects. Significant differences (p=0.003) between the groups were found for Actinomyces odontolyticus/meyeri and Actinomyces israelii which were associated with periodontitis patients. Actinomyces dentalis was found in higher percentage (p=0.015) in the periodontitis group. Actinomyces gerencseriae and Actinomyces massiliensis were significantly more often found supragingivally than subgingivally (p=0.004, p=0.022, respectively) in the periodontitis group. Whether some Actinomyces species, definitely important plaque formers, are actively involved in the pathogenicity of chronic periodontitis needs further investigation.

Ultrastructure and morphology of biofilms on thermoplastic orthodontic appliances in 'fast' and 'slow' plaque formers

The aim of this study was to investigate the morphological features and distribution of biofilms on Invisalign orthodontic appliances, in a sample of 'slow' and 'fast' plaque formers using scanning electron microscopy (SEM). Fifty-six Chinese male/female volunteers (aged 19-39 years) were screened for their plaque-forming rate using the plaque percentage index (PPI) coupled with digital photography and computer-based image analysis, after a period of 48 hours of abstinence from oral hygiene procedures. Eleven volunteers (seven males/four females) representing the lowest and highest ends of the plaque formation spectrum were chosen as slow and fast plaque formers, respectively. The subjects wore a full-coverage splint appliance, in which four tiles of Invisalign material were embedded. These tiles were collected at intervals of 1, 3, 6, 12, 24, and 48 hours, as well as 3, 7, and 14 days, immediately fixed in 10 per cent paraformaldehyde in 0.2 M cacodylate buffer solution and prepared for SEM. The surface configuration of the Invisalign appliance was visualized, as well as the chronological pattern of biofilm formation. Significance between fast and slow plaque formers was determined using a Student's t-test. Colonization appeared to centre initially on the raised edges or textured surfaces of the appliance, and initial adhesion was quicker and more abundant in the fast plaque-forming group. In the later stages of biofilm development, both groups showed no discernible differences in biofilm accrual on the surfaces, but the fast group displayed a more complex biofilm structure. More recessed and sheltered areas of the appliance, such as the cusp tips and attachment dimples, harboured more biofilm than the flat surfaces. Hence, it seems that the novel Invisialign orthodontic appliance is a useful tool to investigate the features of biofilm formation in time-course studies.

New aspects and new concepts of maintaining “microbiological” health

The human mouth is host to a diverse collection of microorganisms including bacteria, viruses, fungi and protozoa. Recent advances using molecular methods for the analysis of complex bacterial communities have demonstrated the richness of the oral bacterial biota and the presence of numerous previously undescribed lineages. Dental plaque forms in a structured way with pioneer species able to colonise pellicle-coated enamel followed by secondary plaque formers such as Fusobacterium nucleatum that have the capability of coaggregating with a range of other genera and species. The mature plaque biofilm has many features of multicellular organisms with the constituent organisms cooperating to make nutrients available and resist environmental stresses, and communicating to regulate their overall numbers. Control of the oral microbiota to prevent disease has conventionally been by mechanical means augmented with toothpastes and mouthrinses, but improved knowledge of oral microbial ecology is allowing the development of preand pro-biotic approaches. Other possibilities include interference with the plaque formation process and the perturbation of bacterial communication networks.

An in vivo replica study of microbial colonization in "rapid" and "slow" dental plaque formers.

The aim of the present study was to investigate the morphological features of supragingival plaque development in "rapid" and "slow" plaque formers using the replica technique. Forty-nine healthy volunteers were screened for their plaque formation rate after 3 days of oral hygiene abstinence using the plaque index (PI). Five subjects with the highest mean PI and six with the lowest mean PI were selected as "rapid" and "slow" plaque formers, respectively. After a series of thorough prophylaxis and oral hygiene instruction, all subjects went through a 14-day period of no oral hygiene to allow undisturbed plaque formation. Light body silicone elastomeric impressions were taken for the upper right central incisor of each subject on day-0, 1, 3, 7, and 14. Positive replicas were then poured from the impressions using epoxy resin and observed under a scanning electron microscope. A more complex supragingival plaque structure was seen in the day-1 and day-3 replicas of the "rapid" group compared to the "slow" group. From day-7 to day-14, during the maturation period of supragingival plaque, no discernible differences were noted between the two groups. Quantitatively, the percentage of plaque coverage on the tooth surfaces was higher in the "rapid" group than in the "slow" group in day-1 and day-3 replicas. These observations indicate that the morphological and topographical features of supragingival plaque in "slow" and "rapid" plaque formers differ, especially in the early developmental phase.

Home use oral hygiene product trials; timing of the last brushing before scoring; an assessment of variation

Home use studies to evaluate oral hygiene products often standardise the time lag between plaque scoring and the previous toothbrushing. Most protocols have favoured an evening before brushing regimen, but the rationale and even the validity of this approach has not been evaluated. In this study, a group of 30 adult subjects participated in a 4-period randomised single-blind crossover evaluation of within-subjects and between-subjects variation in plaque levels after two different brushing times. Thus, on 2 occasions, plaque was scored after an "evening before" brushing and on the other 2 occasions plaque was scored after a "morning before" brushing. As expected, mean plaque levels were lower after morning brushing, but only by 11%. There was little difference for lingual plaque (4%) but a greater difference for buccal plaque (18%). Comparisons for within-subjects variation, which ideally should be low, favoured morning brushing but differences were small and not significant. Comparisons for between subjects variation, which ideally should be high to permit discrimination between high and low plaque formers, also favoured morning brushing but were only significant for lingual plaque. Intraclass correlation coefficients of reliability revealed that overall repeatability was high for both morning and evening regimens; marginally favouring morning brushing. Analyses using all four scores per subject disregarding timing of brushing increased within subjects variation and decreased repeatability, particularly for buccal plaque. In conclusion, the data support the concept of standardising the time between plaque scoring and the previous tooth-brushing. There were no clear statistically significant grounds for preferring one brushing regimen to the other, however the data favoured the morning brushing.

The Tum Protein of Coliphage 186 Is an Antirepressor

The tum gene of coliphage 186, encoded on a LexA controlled operon, is essential for UV induction of a 186 prophage. Primer extension analysis is used to confirm that Tum is the sole phage function required for prophage induction and that it acts against the maintenance repressor, CI, to relieve repression of the lytic promoters, pR and pB, and thereby bring about lytic development. In vitro experiments with purified proteins demonstrate that Tum prevents CI binding to its operator sites. Tum does not compete with CI for binding sites on DNA, and unlike RecA mediated induction of lambda prophage, the action of Tum on CI is reversible. Mechanisms by which Tum may act against CI are discussed.

Scanning electron microscopy of microbial colonization of ‘rapid’ and ‘slow’ dental-plaque formers in vivo

The aim was to investigate the morphological features of supragingival plaque development in ‘rapid’ and ‘slow’ plaque formers using scanning electron microscopy (SEM). Forty-nine healthy volunteers were screened for their plaque-formation rate after 3 days of oral hygiene abstinence using the Plaque Index (PI). Five with the highest and six with the lowest mean PI were selected as ‘rapid’ and ‘slow’ plaque formers, respectively. Six enamel blocks measuring 2 × 2 × 1 mm were bonded onto the buccal surfaces of the upper left canine, premolars and first molar of each selected participant after a series of prophylaxes and oral hygiene instruction to ensure clinical gingival health. A 14-day period with no oral hygiene began thereafter. An enamel block was removed at 3 hr, 6 hr, 1 day, 3 days, 7 days and 14 days from each individual, fixed, and processed for SEM. Quantitatively, more bacteria were observed on the 3-hr to 1-day enamel blocks of the ‘rapid’ plaque formers. The day-1 specimens of the ‘rapid’ group showed a more complex supragingival plaque structure than those of the ‘slow’ group. From days 3 to 14, during the maturation period of supragingival plaque, there were no discernible differences between the two groups except that intermicrobial matrix was more prominent in the ‘rapid’ group. In general, the development of supragingival plaque followed known patterns. These observations indicate that morphological features of supragingival plaque in ‘slow’ and ‘rapid’ plaque formers differ, especially in the early developmental phases.

Effect of delmopinol hydrochloride mouthrinse on plaque formation and gingivitis in “rapid” and “slow” plaque formers

The aim of the present study was to investigate differences in the plaque and gingivitis inhibiting effect of delmopinol rinsing between "rapid" and "slow" plaque formers. 23 subjects (12 "rapid" and 11 "slow" plaque formers) were selected from 71 healthy young adults. The selection was based on the plaque index on the buccal surfaces of all premolars and 1st molars after 3-days without plaque control. The 23 subjects were randomly assigned into 3 groups with different mouthrinses, i.e., 0.1% delmopinol, 0.2% delmopinol, and placebo. The study was double-blind with parallel design between the "rapid" and "slow" plaque formers and cross-over design between 2 active periods and a placebo period. Each rinsing period lasted for 5 days. During the 3 test periods, the subjects refrained from all oral hygiene and rinsed 2x daily with either one of the 3 solutions. Gingival crevicular fluid (GCF) was collected from buccal surfaces of upper canines and premolars and bleeding on probing (BOP) recorded at 6 sites around each tooth before and after each test period. Plaque assessment, including plaque index (PI) and standardized color slides for planimetric analyses obtained from the canines and premolars, were only recorded after each test period. Results showed that the mean PI and planimetry values for both the "rapid" and "slow" plaque formers were lower than the placebo, for either the 0.1% or the 0.2% delmopinol mouthrinse. The differences between the" rapid" and "slow" plaque formers were not statistically significant. There was a small reduction in BOP in both groups for the delmopinol periods, as against a slight increase in the placebo period; the difference between the placebo group and the 2 groups of plaque formers was not statistically significant (p>0.6 for both 0.1% and 0.2% delmopinol). Results suggested that both 0.1% and 0.2% delmopinol reduce plaque formation and gingivitis to a similar extent in subjects with extreme rates of plaque formation.

Predominant cultivable supragingival plaque in Chinese "rapid" and "slow" plaque formers.

The aim of this study was to compare the predominant cultivable bacterial flora in supragingival plaque samples of Chinese "rapid" and "slow" plaque formers, using the experimental gingivitis model. 11 Chinese subjects (5 "rapid" and 6 "slow" plaque formers) were selected from 49 healthy young adults. The selection was based on the plaque index on the buccal surfaces of all the canines, premolars and 1st molars after 3-days without plaque control. The 11 subjects began a 14-day on oral hygiene period after prophylaxis. Plaque samples were collected on day 1, day 3, day 7 and day 14 from the buccal surface of upper right canine, 2nd premolar, 1st premolar and 1st molar, respectively. The samples were then dispensed into a tryptic soy broth, and cultured anaerobically to obtain pure isolates which were then identified by conventional means. Results showed similar trends in plaque development in the two groups; gram-positive bacteria were the predominant cultivable species ("rapid": 71-37%; "slow": 53-63%) and gram-negative species increased in proportion to the plaque age ("rapid": 9-47%, "slow": 13-28%). "Rapid" plaque formers showed a statistically significant higher percentage of gram-negative rods (38%) than the "slow" group (17%) in the 14-day samples. The difference in the proportions of other groups of bacteria between the "rapid" and "slow" plaque formers were also found to be statistically significant using the MANOVA test (p = 0.0162). Most of the cultivable gram-negative rods belonged to Fusobacterium and Capnocytophaga species. Besides the quantitative difference observed clinically, there seems to be a qualitative difference in the cultivable flora between subjects with different rates of plaque formation.

Lactotrope subtypes are differentially responsive to calcium channel blockers

Pituitary cultures from adult rats contain two subtypes of prolactin (PRL) cells, small-plaque (SP) and large-plaque (LP) lactotropes, which exhibit distinct rates of basal secretion and thereby form PRL plaques of different sizes in reverse hemolytic plaque assay experiments. In the present study, we have used plaque assays to examine the effects of ω-conotoxin (ω-CgTx) and nifedipine, which block Ca 2+ entry through high voltage-activated (HVA) channels in the plasma membrane, on basal PRL secretion from single male rat lactotropes. We found that ω-CgTx, like nifedipine, is a potent inhibitor of PRL secretion. In addition, we observed that both drugs decrease the number of cells forming large PRL plaques, while promoting a comparable increase in the abundance of small plaque formers. The results indicate that blocking the HVA Ca channels preferentially suppresses PRL release from LP lactotropes, and suggest that the inhibited PRL secretors tend to behave functionally as SP lactotropes.

Morphopoietic switch mutations of bacteriophage P2

During the growth of bacteriophage P4, for which the genome of bacteriophage P2 is needed as helper, the decision whether to make large, P2 size, heads or small, P4 size, heads depends on the size-directing function of P4's sid gene and on P2's “ sid responsiveness.” P2 mutants (=P2 sir) impaired in their response to P4's sid function are readily obtainable as one class of P2 plaque formers selected on certain P4 cl plasmid lysogens. We describe nine P2 sir mutants of independent origin. For eight we could assign their sir mutation to P2 gene N, which encodes the major capsid protein. DNA sequencing indicated an open reading frame of 357 codons for gene N and showed these sir mutations to affect only four codons within a 38-codon segment in the middle of N. Seven mutations are missense mutations (three of them identical); one is a deletion of one codon. There seems to be a correlation between the phenotypic “strength” of the sir mutations and the type of amino acid replacement by missense mutations. Although the weakest mutation, sir7, could not yet be assigned to any P2 gene, it appears clear from this work that P2's N gene product is the major (or only) target of P4's Sid gene function.

Relative Importance of Newly Synthesized and Stored Hormone to Basal Secretion by Growth Hormone and Prolactin Cells*

It is generally accepted that under basal conditions there is preferential release of newly synthesized hormone by a number of endocrine cell type, including those that secrete GH or PRL. However, the cellular basis for this phenomenon along with the relative contribution of stored hormone to basal secretion has yet to be clearly established. In the present study, we employed reverse hemolytic plaque assays to monitor basal and stimulated release of GH and PRL from individual cells in which de novo protein synthesis had been blocked. Monodispersed pituitaries from adult male rats were cultured for 21 h in the absence or presence of maximally effective doses of puromycin (100 microM) or cycloheximide (36 microM) and were then subjected to separate plaque assays for GH or PRL. Treatment with puromycin reduced the percentage of GH or PRL secretors (plaque formers) by about half. Coincubation with stimulatory secretagogues did not increase the percentages of GH or PRL secretors in control cultures, but returned the proportion in puromycin-treated cells to normal, demonstrating that cells which failed to secrete basally could still release hormone from their stored pools when stimulated. Very similar results were obtained when these experiments were repeated with cycloheximide. Taken together, these results demonstrate that only a fraction of the cells that release GH or PRL are dependent upon newly synthesized hormone for basal secretion; the remainder appear capable of mobilizing stored hormone for this purpose even in the absence of stimulation.

Influence of saliva from 'heavy' and 'light' plaque formers on the colloidal stability of bacterial suspensions.

The influence of parotid saliva on the colloid stability of suspensions of Streptococcus sanguis and S. salivarius was studied in groups of previously identified 'heavy' and 'light' plaque formers. For S. sanguis it was observed that addition of parotid saliva from light plaque formers had more pronounced negative influence on the colloid stability than addition of such saliva from heavy plaque formers. No differences were observed for S. salivarius. The results indicate that saliva and bacteria might be regarded as a biological colloidal system and that the individual rate of plaque formation can perhaps be partially related to the colloid-chemical properties of bacteria and saliva.

Effects of DNA heterologies on bacteriophage lambda packaging.

We have examined the impact of DNA heterologies on the packaging of lambda DNA in vitro. Heterology-containing DNA molecules were constructed by denaturing and reannealing a mixture of DNA from cI+ phage and DNA front phage carrying small insertion or deletion mutations in the cI gene. We found that molecules with heterologies of up to 19 base pairs (bp) can be packaged as viable heterozygous phage with approximately the same efficiency as molecules with a base pair mismatch. In contrast, with a heterology of 26-bp heterozygous plaque formers are rare. In principle, the absence of cI heterozygotes among packaged phage may be due either to a failure to encapsulate the DNA or a failure to inject the packaged DNA on infection. Southern blot analysis of DNA isolated from packaged phage indicates that DNA harboring a 26-bp heterology is almost completely absent in packaged phage. Thus, an upper limit has been established for the size of heterology that can be accommodated by the packaging apparatus The size of the connector portal could be the basis for this limit.

A novel P22 prophage in Salmonella typhimurium.

Under several sets of conditions, all of which seem to perturb purine metabolism, Salmonella typhimurium releases a variety of phages which were not known to be present in the strain. These cryptic phages are not induced by UV irradiation. Furthermore, the induction process does not require a functional recA gene product. While phages of several phenotypic classes have been recovered, including both turbid and clear plaque formers, all appear to be variants of P22 because all show DNA restriction patterns indistinguishable from that of P22. The variety of types suggests that the cryptic prophage is mutagenized as a consequence of the induction process. All the temperature phages tested are capable of transducing a variety of chromosomal markers with high efficiency. The phages induced in this novel way are capable of forming plaques on the strains that gave rise to them. Since the strains releasing phage are not immune to P22, the parental lysogens must not express immunity and the phage must be held in a cryptic state by a novel mechanism. The released phage possess an intact P22 immunity system because many can form standard immune lysogens after reinfection of Salmonella. These results raise the possibility that Salmonella typhimurium harbors cryptic phages that are subject to a novel system of global control related to purine metabolism. Preliminary evidence suggests that the regulation system may involve DNA modification.

UV-inducible DNA repair in Acinetobacter calcoaceticus

Bacterial mutation frequency after UV irradiation and phage mutation frequency under conditions of W-reactivation were determined in A. calcoaceticus. With the exception of streptomycin resistance, there was no increase in the frequency of the assayed merkers above the background level. The increased survival of phage during W-reactivation was not followed by an increase in the frequency of mutation from turbid to clear plaque formers among phage survivors. The findings suggested that the UV-inducible repair pathway in A. calcoaceticus was error free. Post-irradiation incubation of UV-treated culture before phage infection resulted in a further increase of W-reactivation. As chloramphenicol inhibited this response, it was concluded that de novo protein synthesis was involved in the UV-inducible repair pathway in A. calcoaceticus.

Biophysical and microbiologic studies of “heavy” and “light” plaque formers

In order to trace factors possibly influencing initial bacterial colonization of tooth surfaces, a multiple linear regression analysis was performed on the present results as well as on those from certain clinical and biochemical studies previously performed in the same subjects. No statistically significant differences were observed between the two groups of plaque formers for the individual parameters. The multiple regression analysis showed, however, that the calculated work of adhesion for two polar and one non polar test liquid, the rate of unstimulated salivary secretion, morphologic differences in the dento-gingival areas of maxillary premolars and saliva-induced aggregation of S. mitior were crucial for about 90% of the variation in the number of CFU colonizing tooth surfaces after both 2 and 6 h. Both electrostatic and van der Waal's forces, as well as salivary flow conditions could thus have influenced the initial attachment of bacteria to the test surfaces. It is concluded that saliva including its suspended microorganisms could be looked upon as being a colloidal biological system, and if so, the initial bacterial colonization of tooth surfaces should follow known, general biophysical laws.

Competitive displacement of mutans streptococci and inhibition of tooth decay by Streptococcus salivarius TOVE-R.

The ability of Streptococcus salivarius TOVE-R to displace virulent representatives of the most prevalent human mutans streptococci from the teeth of rats, and thereby to inhibit caries, was studied. Streptococcus mutans 10449S- or Streptococcus sobrinus 6715-13WT-infected specific-pathogen-free rats consuming a high-sucrose diet were inoculated by TOVE-R. The infectants were differentially recovered from swabs of the teeth over the time course of infection and from sonically treated material of extracted teeth and excised tongues. Despite initial colonization of the teeth by the mutans streptococci, TOVE-R colonized the teeth, unlike other essentially nonvirulent plaque formers already described. It did not colonize the tongues of the rats. TOVE-R emerged and persisted as a prominent member of the plaque ecology. There was an associated decline in the mutans streptococci on the teeth, and this decline was associated with significant inhibition of the caries component attributable to 10449S infection (56%) and to 6715-13WT infection (52%). TOVE-R did not reliably inhibit the component of fissure caries attributable to the nonmutans indigenous flora of the rats. TOVE-R itself induced no detectable decay. The data suggest the potential therapeutic utility of TOVE-R to inhibit caries by displacement of mutans streptococci from the teeth. These results supplement the already reported ability of TOVE-R to preempt initial colonization of teeth by the mutans streptococci.