Evaluation of plant and fungal extracts for their potential antigingivitis and anticaries activity.

The link between diet and health has lead to the promotion of functional foods which can enhance health. In this study, the oral health benefits of a number of food homogenates and high molecular mass and low molecular mass fractions were investigated. A comprehensive range of assays were performed to assess the action of these foods on the development of gingivitis and caries using bacterial species associated with these diseases. Both antigingivitis and anticaries effects were investigated by assays examining the prevention of biofilm formation and coaggregation, disruption of preexisting biofilms, and the foods' antibacterial effects. Assays investigating interactions with gingival epithelial cells and cytokine production were carried out to assess the foods' anti- gingivitis properties. Anti-caries properties such as interactions with hydroxyapatite, disruption of signal transduction, and the inhibition of acid production were investigated. The mushroom and chicory homogenates and low molecular mass fractions show promise as anti-caries and anti-gingivitis agents, and further testing and clinical trials will need to be performed to evaluate their true effectiveness in humans.

Levels of periodontal pathogens in neonatal gastric aspirates and possible maternal sites of origin.

Maternal periodontal infection has been recognized as a risk factor for preterm and low birthweight infants. It is suspected that pathogens causing periodontal disease may translocate to the amniotic cavity and so contribute to triggering an adverse pregnancy outcome. This study aimed to determine levels and proportions of periodontal bacteria in neonatal gastric aspirates obtained from complicated pregnancies and the respective maternal oral and vaginal samples using a quantitative polymerase chain reaction approach, and also to determine the origin of the neonate's bacteria by sequence comparisons between the three sites. Aggregatibacter actinomycetemcomitans and Tannerella forsythia were not observed in the neonates or in the women's vaginas. Interestingly, Porphyromonas gingivalis was identified in the neonates in two samples (2.98E+02 and 1.75E+02 cells ml(-1)) and in association with Fusobacterium nucleatum, which was observed at high prevalence (10%) and at high levels reaching up to 2.32E+03 cells ml(-1). Although F. nucleatum was also present in the vaginal samples, the results demonstrated that the neonatal strains were more likely to originate from the mother's oral cavity than to be vaginal strains.

An investigation of a potential confounder in ex vivo microbiological studies--the bulk flow of fluid through apical foramina during tooth extraction.

AIMS: To investigate the factors affecting bulk flow of dye and bacterial suspensions into and out of apical foramina during simulated tooth extraction, using an ex vivo model. METHODOLOGY: Sixty extracted, single-rooted, human teeth were accessed, root canals located and in 50 the pulps dissolved; 10 teeth with attached periapical lesions were preserved. The size of apical foramina was determined digitally. The teeth were mounted in vials with polyvinylsiloxane impression material. Part 1: different dyes were inoculated in the coronal half of root canals or cervical 'gingival' margin, respectively, in separate experiments using the same teeth. Tooth extraction movements were simulated and apical penetration of the dye solutions with and without coronal restorations were examined in each case (20 teeth re-used 4 × ). Part 2: the same procedures were repeated on 30 more teeth but using a standard inoculum of Acidovorax sp. Part 3: 10 teeth with attached periapical lesions were inoculated with Acidovorax sp. in the absence of coronal restorations. Bacterial leakage into the periapical lesions was assessed. RESULTS: Coronal restorations significantly reduced the flow of dyes (P=0.002) or bacterial suspension (P=0.001) out of the canals and bacterial suspension into (P=0.02) the canals during simulated tooth extraction. The 'size of apical foramina' were positively correlated with passage of bacterial suspension out of the canal (P=0.04) and from the gingival trough into the canal (P=0.008), in the presence of a coronal restoration. CONCLUSIONS: The presence of coronal restorations, the size of apical foramina and presence of native canal contents with attached periapical lesions, all influenced fluid flow into and out of canals during simulated tooth extraction movements.

Disease severity associated with presence in subgingival plaque of Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, and Tannerella forsythia, singly or in combination, as detected by nested multiplex PCR.

This study used a nested multiplex PCR method to detect three periodontal pathogens in subgingival plaque collected before treatment and at 2 and 6 months posttreatment from 107 patients with severe, generalized periodontitis. The proportions of the patients who harbored these bacteria before periodontal treatment were as follows: Tannerella forsythia, 81%; Porphyromonas gingivalis, 78%; and Aggregatibacter (formerly Actinobacillus) actinomycetemcomitans, 47%. At 2 months posttreatment there was a significant reduction in the numbers of patients harboring P. gingivalis (46%; P < 0.001) or T. forsythia (63%; P = 0.043) but not A. actinomycetemcomitans (50%) compared to pretreatment data. At 6 months posttreatment, significantly fewer patients harbored P. gingivalis (43%; P < 0.001); A. actinomycetemcomitans, (31%; P = 0.025), or T. forsythia (63%; P = 0.030). Interestingly, at baseline and at 2 months posttherapy, subjects who harbored only a single pathogen had a greater level of periodontal disease than subjects who harbored two, or all three, of these periodontal pathogens. These data suggest that a reduction in the number of species present may be associated with an increase in the severity of periodontal diseases.

Use of quantitative PCR and culture methods to characterize ecological flux in bacterial biofilms.

An in vitro model of supragingival plaque associated with gingivitis was characterized by traditional culture techniques, comparative 16S rRNA gene sequencing of isolates, and quantitative PCR (QPCR). Actinomyces naeslundii, Prevotella spp., and Porphyromonas gingivalis increased under conditions emulating gingivitis. Gram-negative species and total bacteria were dramatically underestimated by culture compared to the estimates obtained by QPCR.

Determination of the genetic support for tet(W) in oral bacteria.

The DNA sequence flanking a tet(W) gene in an oral Rothia sp. was determined. The gene was linked to two different transposases, and these were flanked by two almost identical mef (macrolide efflux) genes. This structure was found in 4 out of 20 tet(W)-containing oral bacteria investigated.

Modeling shifts in microbial populations associated with health or disease.

Stable microbial communities associated with health can be disrupted by altered environmental conditions. Periodontal diseases are associated with changes in the resident oral microflora. For example, as gingivitis develops, a key change in the microbial composition of dental plaque is the ascendancy of Actinomyces spp. and gram-negative rods at the expense of Streptococcus spp. We describe the use of an in vitro model to replicate this population shift, first with a dual-species model (Actinomyces naeslundii and Streptococcus sobrinus) and then using a microcosm model of dental plaque. The population shift was induced by environmental changes associated with gingivitis, first by the addition of artificial gingival crevicular fluid and then by a switch to a microaerophilic atmosphere. In addition to the observed population shifts, confocal laser scanning microscopy also revealed structural changes and differences in the distribution of viable and nonviable bacteria associated with the change in environmental conditions. This model provides an appropriate system for the further understanding of microbial population shifts associated with gingivitis and for the testing of, for example, antimicrobial agents.

Duration, prevalence and intensity of bacteraemia after dental extractions in children.

OBJECTIVE: To investigate the duration, prevalence and intensity of bacteraemia after dental extractions in children by comparing within-patient bacteraemia before and after dental extraction. METHODS: Children were randomly allocated to one of 10 postprocedure time groups from 10 s to 60 min. The differences between intensity and prevalence of the bacteraemia at each time after extractions were used to estimate the duration of the bacteraemia. After attainment of general anaesthesia, pre-extraction and postextraction blood samples were processed by broth culture and lysis filtration to isolate and quantify bacteria present in the patients' blood. RESULTS: 500 subjects between 3 and 16 years old were recruited. The estimated duration of bacteraemia was about 11 min. CONCLUSIONS: The duration of bacteraemia after dental extractions is less than previously thought. This has implications for the interpretation of odontogenic bacteraemia studies.

An in vitro evaluation of the ability of ozone to kill a strain of Enterococcus faecalis.

AIM: To evaluate the potential of ozone as an antibacterial agent using Enterococcus faecalis as the test species. METHODOLOGY: Ozone was produced by a custom-made bench top generator and its solubility in water determined by ultraviolet (258 nm) spectrophotometric analysis of solutions through which ozone was sparged for various time-periods. The antibacterial efficacy of ozone was tested against both broth and biofilm cultures. Ozone was sparged for 30, 60, 120 and 240 s, through overnight broth cultures of a strain of E. faecalis (E78.2) and compared with those that were centrifuged, washed and resuspended in water. Enterococcus faecalis (E78.2) biofilms were grown on cellulose nitrate membrane filters for 48 h and suspended in water through which ozone gas was sparged with stirring for 60, 120 and 240 s in a standard fashion. In a separate test, biofilms were also exposed to gaseous ozone. Sodium hypochlorite (NaOCl) was used as a positive control. All experiments were repeated four times. RESULTS: There were significant (P < 0.05) reductions of bacteria in the unwashed (2 log(10) reductions) and washed (5 log(10) reductions) broth cultures following 240 s applications. Biofilms incubated for 240 s with ozonated water showed no significant reduction in cell viability attributable to ozone alone, whereas with NaOCl no viable cells were detected over the same time. Gaseous ozone applied for 300 s had no effect on these biofilms. CONCLUSIONS: Ozone had an antibacterial effect on planktonic E. faecalis cells and those suspended in fluid, but little effect when embedded in biofilms. Its antibacterial efficacy was not comparable with that of NaOCl under the test conditions used.

Effectiveness of electrochemically activated water as an irrigant in an infected tooth model.

AIM: To test the effectiveness of electrochemically activated aqueous solutions in the debridement of Enterococcus faecalis biofilms in root canals of extracted teeth. METHODOLOGY: Extracted, human, single-rooted teeth (198) assembled into 11 sets (n = 18) with matching anatomical characteristics were randomly assigned to eight experimental groups. After decoronation, the root canals were prepared to a standard size. Enterococcus faecalis biofilms were grown in the root canals of autoclaved, individually mounted teeth over 48 h. Electrolysed saline collected as anolyte at the anode and catholyte at the cathode were the test agents. The four ultrasonication and four without ultrasonication irrigant groups included: neutral anolyte (NA) (pH 6.5), acidic anolyte (AA) (pH 3.0), catholyte (C) (pH 11.5) and C alternated with neutral anolyte (C/NA). Phosphate-buffered saline (PBS) with and without ultrasonication formed negative and NaOCl (3%) positive control groups. After irrigation, root canal samples were serially diluted, cultured and enumerated. The data were analysed as ratios of residual colony-forming units (CFUs) in PBS versus the test irrigants and using multivariate regression. RESULTS: The NA and NA (ultrasonicated, U), C/NA and AA (U) groups had significantly (alpha = 0.05) less and C (U) and C/NA (U) significantly (alpha = 0.05) more bacteria (CFUs mL(-1)) compared with their respective PBS controls. Ultrasonicated C/NA had significantly (alpha = 0.05) higher CFU counts than the nonultrasonicated solution. Other comparisons between ultrasonic and nonultrasonic groups were not significant. Of the nonultrasonicated groups, C/NA and NA were most effective, whilst of the ultrasonicated groups, AA and NA were most effective. None of these was as effective as 3% NaOCl. CONCLUSIONS: All but two groups (AA and C) were significantly different from their PBS controls. There was a significant difference between the C/NA groups with and without ultrasonication but not between other combinations. NA (U) and AA (U) were the most effective test solutions but NaOCl (3%) gave by far the highest bacterial kills.

In vitro modeling of dental water line contamination and decontamination.

The contamination of dental unit water lines (DUWL) is an emerging concern in dentistry. The aim of this study was to use an in vitro DUWL to model microbial contamination and evaluate the decontamination efficacy of tetraacetylethylenediamine (TAED) solutions. A DUWL biofilm model used to simulate clinical conditions was used to generate a range of biofilms in DUWL. Three distinct biofilms were generated: (1) biofilm from water, (2) biofilm from a mix of water + contaminating human commensal bacteria, (3) biofilm from water with contaminating oral bacteria added after biofilm formed. The contaminating oral species used were Streptococcus oralis, Enterococcus faecalis and Staphylococcus aureus. Decontamination by simple water flushing or flushing with TAED was evaluated (2, 5 and 10 min intervals). The DUWL tubes were split and samples were plated onto a range of media, incubated and bacteria enumerated. Water flushing did not reduce the number of microorganisms detected. Bacteria were not detected from any of the TAED sampling points for any of the biofilm types tested. Interestingly, if contamination was introduced to new DUWL along with the waterborne species a biofilm was formed containing only the waterborne species. If however, an existing biofilm was present before the introduction of "contaminating" bacteria then these could be detected in the biofilm. This implies that if the DUWL are new or satisfactorily cleaned on a regular basis then the associated cross-contamination aspects are reduced. In conclusion, TAED provides effective control for DUWL biofilms.

Genetic basis of erythromycin resistance in oral bacteria.

We determined the prevalence of erythromycin-resistant bacteria in the oral cavity and identified mef and erm(B) as the most common resistance determinants. In addition, we demonstrate the genetic linkage, on various Tn1545-like conjugative transposons, between erythromycin and tetracycline resistance in a number of isolates.

Novel tetracycline resistance determinant from the oral metagenome.

A major drawback of most studies on how bacteria become resistant to antibiotics is that they concentrate mainly on bacteria that can be cultivated in the laboratory. In the present study, we cloned part of the oral metagenome and isolated a novel tetracycline resistance gene, tet(37), which inactivates tetracycline.

Prevalence of tetracycline resistance genes in oral bacteria.

Tetracycline is a broad-spectrum antibiotic used in humans, animals, and aquaculture; therefore, many bacteria from different ecosystems are exposed to this antibiotic. In order to determine the genetic basis for resistance to tetracycline in bacteria from the oral cavity, saliva and dental plaque samples were obtained from 20 healthy adults who had not taken antibiotics during the previous 3 months. The samples were screened for the presence of bacteria resistant to tetracycline, and the tetracycline resistance genes in these isolates were identified by multiplex PCR and DNA sequencing. Tetracycline-resistant bacteria constituted an average of 11% of the total cultivable oral microflora. A representative 105 tetracycline-resistant isolates from the 20 samples were investigated; most of the isolates carried tetracycline resistance genes encoding a ribosomal protection protein. The most common tet gene identified was tet(M), which was found in 79% of all the isolates. The second most common gene identified was tet(W), which was found in 21% of all the isolates, followed by tet(O) and tet(Q) (10.5 and 9.5% of the isolates, respectively) and then tet(S) (2.8% of the isolates). Tetracycline resistance genes encoding an efflux protein were detected in 4.8% of all the tetracycline-resistant isolates; 2.8% of the isolates had tet(L) and 1% carried tet(A) and tet(K) each. The results have shown that a variety of tetracycline resistance genes are present in the oral microflora of healthy adults. This is the first report of tet(W) in oral bacteria and the first report to show that tet(O), tet(Q), tet(A), and tet(S) can be found in some oral species.

Characterization of in vitro oral bacterial biofilms by traditional and molecular methods.

The aim of this study was to compare culture-based bacterial isolation methods with direct amplification and cloning of 16S rRNA genes from oral biofilms grown in an in vitro model. The model used was a constant depth film fermentor which was inoculated with pooled human saliva. The use of culture techniques and cloning resulted in the identification of 36 different bacterial species from the saliva inoculum and from the biofilms. Of these, only five were detected solely by molecular methods. Three taxa were detected which, according to the databases, were unidentified. Using the molecular methods of detection, differences in the number of species observed were found using different 16S rRNA gene primers and numbers of PCR cycles. We have shown that microcosm supragingival plaque biofilms grown in a fermentor consisted of a community most of the members of which could be cultivated on laboratory media.

Prevalence, proportions, and identities of antibiotic-resistant bacteria in the oral microflora of healthy children.

The aims of this study were to determine the prevalence, proportions and identities of oral bacteria resistant to six antibiotics in 35 children (4-5 years old) who had not received antibiotics during the previous 3 months. Ampicillin-, penicillin-, erythromycin-, and tetracycline-resistant bacteria were harbored by 35 (100%), 34 (97%), 35 (100%), and 34 (97%) children, respectively. None of the children harbored metronidazole-resistant anaerobic bacteria or Gram-positive vancomycin-resistant bacteria. The median percentage of the oral microflora resistant to each of the antibiotics was ampicillin 1% (range 0.1-23), erythromycin 13% (1-45), penicillin 1% (0-14), and tetracycline 2% (0-88). A total of 432 antibiotic-resistant isolates were recovered that comprised 18 genera and 47 species. Ampicillin resistance was widely distributed throughout different genera and species, whereas tetracycline resistance was predominately found in the streptococci. Multiresistant bacteria were frequently isolated with 28% of isolates exhibiting resistance to two or more antibiotics. Veillonella spp., traditionally considered susceptible to penicillin and ampicillin, were found frequently to be resistant to these two antibiotics. This study demonstrates that a diverse collection of antibiotic-resistant pathogenic, opportunistic, and nonpathogenic bacteria can be readily isolated from, and in some subjects dominate, the oral microflora of primary school children in the absence of recently administered antibiotics.

Prevalence of yeasts in saliva and root canals of teeth associated with apical periodontitis.

AIMS: To determine: (i) the relative prevalence and diversity of yeasts in salivary and root canal samples from the same patients; and (ii) the clinical factors associated with their presence in saliva and root canals. METHODOLOGY: Sixty root canal samples from teeth associated apical periodontitis and the corresponding whole unstimulated saliva samples were obtained from 55 patients. The medical history including antibiotic therapy and clinical/radiographic data on the teeth were recorded. The samples were serially diluted and cultured on yeast & fungi-selective sabouraud dextrose agar. Isolates were characterized and speciated by the germ tube formation test, hyphal morphology and a commercial biochemical test kit (Rapid ID32C(R) system). RESULTS: Twenty-three yeast isolates were recovered from 19 saliva samples and eight isolates from six root canal samples. Candida albicans (17/23 & 3/8) and Rodotorula mucilaginosa (2/23 & 4/8) were the most prevalent isolates from saliva and root canal samples. It was significantly (13.8 times) more probable that yeasts would be recovered from root canals when they were also present in the saliva (P = 0.021). The effect of coronal restoration leakage (P = 0.08) and previous root canal treatment (P = 0.123) were equivocal. The history of antibiotic therapy had no association with the presence of yeasts in saliva (OR = 1.1). CONCLUSIONS: Yeasts occurred relatively infrequently (10%) in root canals. Their presence in root canals was significantly associated with their presence in saliva. The role of yeasts in the initiation and perpetuation of periapical disease remains to be determined.

Development of an in vitro microassay for glucose quantification in submicrolitre volumes of biological fluid.

Glucose quantification in serum or plasma is traditionally based on a colourimetric enzymatic assay using commercially available assay kits. Sample volumes of blood or serum are usually in the range of a few microlitres to a few millilitres. However, for biological fluids such as gingival crevicular fluid (GCF), which can only be sampled in submicrolitre volumes, such assays have proven unsuitable. The aim of this study was to develop a reliable and reproducible assay for quantifying glucose in submicrolitre samples of GCF. The assay involved modification of a commercially available kit for glucose quantification. Test solutions of (i) serum and (ii) serum with added glucose at known concentrations (range 50-400 mg/dl) were prepared to simulate GCF and GCF enriched with glucose, respectively. Submicrolitre volumes (range 0.2 microl to 0.8 microl) of the test solutions were added to the reagent solution (200 microl) using a Hamilton syringe. The reaction was performed under standard conditions of time and temperature. The colour change was assayed spectrophotometrically at 492 nm. The results showed that this microassay is sufficiently sensitive to detect 50 mg/dl glucose in 0.2 microl of sample and indicate that the accuracy and sensitivity of this assay make it suitable for glucose quantification in submicrolitre volumes of GCF, particularly relevant to investigations of the relationship between diabetes mellitus and chronic inflammatory periodontal disease. In vivo evaluation of this novel microassay was performed using GCF samples taken from periodontally healthy and chronic periodontitis patients. Using non-parametric analysis, the results showed that the assay detected statistically significant differences in glucose concentrations between the two patient groups (p < 0.05). Higher glucose levels were detected at the periodontally diseased sites. For each patient, the GCF-glucose: blood-glucose ratio was calculated. The results show that this ratio was higher in the periodontitis group (1: 2) when compared to the healthy group (1: 9). In conclusion, the results of this investigation have shown that this microassay can quantify glucose in GCF and that GCF-glucose levels are higher at periodontitis sites.

Composition and antibiotic resistance profile of microcosm dental plaques before and after exposure to tetracycline.

The aim of this study was to investigate the effects of tetracycline administration on the viability and antibiotic resistance profiles of microcosm dental plaques. A constant depth film fermenter was used to generate multi-species biofilms, which were grown for 216 h before tetracycline was added. The composition of the microcosm plaques was determined by viable counting on selective and non-selective media. The prevalence of antibiotic-resistant organisms was determined on antibiotic-containing media. Before administration of tetracycline, the biofilms had a total viable anaerobic count of 7 x 10(7) cfu per biofilm. They contained 7% lactobacilli, 19% streptococci and 2% Actinomyces spp. Immediately after pulsing with tetracycline, the composition of the biofilms changed and they consisted of 30% lactobacilli, 1.5% streptococci and 3% Actinomyces spp., with a total anaerobic count of 1 x 10(7) cfu per biofilm. The pre-valence and composition of the antibiotic-resistant microflora changed dramatically after the addition of tetracycline, with the proportion of the microflora displaying resistance to tetracycline increasing from 6% to 45%. Corresponding changes in the proportions of the microflora displaying resistance to other antibiotics were as follows: 5-28% for erythromycin, 1-5% for vancomycin and 0.4-3% for ampicillin. The results of this study have shown that the addition of tetracycline to microcosm dental plaques alters their composition and enriches for bacteria resistant to tetracycline and other unrelated agents.

A new in vitro model for the study of microbial microleakage around dental restorations: a preliminary qualitative evaluation.

AIM: The aim of this study was to develop an in vitro model to replicate microbial microleakage at a tooth/ restoration interface using a constant depth film fermentor (CDFF). METHODOLOGY: Amalgam restorations were placed in machined bovine dentine cylinders and sealed externally with varnish, leaving a 1-mm perimeter exposed around the tooth/restoration interface. The dentine cylinders were housed in a CDFF and 300-microm thick microcosm dental plaques were grown over their exposed surfaces. The biofilms were maintained with a mucin-containing artificial saliva for up to 8 weeks. Cylinders were aseptically removed from the CDFF (at 1, 2, 4, & 8 weeks) and surface-decontaminated with validated protocols prior to splitting and sampling of apposing amalgam and dentine surfaces. Scanning electron microscopy (SEM) was used to ascertain the position and structure of the bacterial aggregates. Bacterial viability was determined by vital staining of the bacteria in situ. RESULTS: At all sampling times, SEM showed cocci, rods and filaments on both amalgam and dentine surfaces; some originated as cascades from the surface biofilm and extended into the tooth/restoration microspace. Vital staining showed the majority of bacteria from both dentine and amalgam surfaces to be viable. CONCLUSION: This preliminary investigation showed that the CDFF may be a valuable tool for the in vitro study of the dynamics of microbial microleakage around dental restorations.