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.

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.

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.

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.

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.

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.

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.

Capnocytophaga gingivalis: effects of glucose concentration on growth and hydrolytic enzyme production.

In chemostat culture, the microaerophilic, CO2 requiring, gingival-plaque-associated bacterium Capnocytophaga gingivalis responded to the addition of glucose (1-6 g I-1) by doubling its growth rate and increasing its biomass yield fivefold. The data suggest that the glucose is catabolized by a fully aerobic route. Rather than repressing hydrolytic enzymes which might be associated with pathogenic properties, glucose enhanced the specific activity of aminopeptidase, trypsin-like protease, acid and alkaline phosphatase and alpha-glucosidase in comparison with a control culture grown in a tryptone/thiamin medium. Thus, the supply of glucose could be of importance in maximizing the pathogenic potential of this organism.

An in vitro evaluation of the antimicrobial efficacy of irrigants on biofilms of root canal isolates.

AIM: The bactericidal effect of four antimicrobial agents was investigated against single-species biofilms derived from a range of root canal isolates. METHODOLOGY: Single-species biofilms of Prevotella intermedia, Peptostreptococcus micros, Streptococcus intermedius, Fusobacterium nucleatum and Enterococcus faecalis were generated on membrane filter discs and subjected to 15 min or 1 h incubation with 5 p.p.m. colloidal silver, 2.25% sodium hypochlorite (NaOCl), 0.2% chlorhexidine, 10% iodine or phosphate buffered saline (PBS) as a control. The antimicrobial activity of the agents was neutralized and the bacterial cells were harvested from the discs by vortexing, serially diluted in reduced transport fluid, plated on fastidious anaerobe agar containing 5% horse blood, incubated anaerobically and colony-forming units calculated. RESULTS: Iodine and NaOCl were more effective than chlorhexidine except against P. micros and P. intermedia where they were all 100% effective. Iodine and NaOCl elicited a 100% kill after 1 h incubation for all strains used. However, after 15 min, they showed differing bactericidal effects depending on the strain. None of the agents were effective against F. nucleatum after 15 min but NaOCl, iodine and chlorhexidine were all effective after 1 h. Colloidal silver was generally ineffective. CONCLUSIONS: The effectiveness of a particular agent was dependent on the nature of the organism in the biofilm and on the contact time. NaOCl was generally the most effective agent tested, followed by iodine. However the clinical efficacy of these agents must be considered in light of the complex root canal anatomy and polymicrobial nature of root canal infections.

Capnocytophaga gingivalis aminopeptidase: a potential virulence factor.

The production and properties of an aminopeptidase from Capnocytophaga gingivalis were studied. C. gingivalis was grown in continuous culture over a range of dilution rates and the cell-bound and extracellular levels of aminopeptidase and trypsin-like protease (TLPase) measured. At high growth rates (0.6 mu rel) TLPase specific activity was low and found exclusively as cell-bound activity; at low growth rates (0.0375 mu rel), specific activity was high and 26% was found as extracellular activity. In contrast, aminopeptidase specific activity was highest at 0.3 mu rel and the ratio of cell-bound to extracellular activity was relatively constant at all growth rates. Only about 5% of the total activity was extracellular. The aminopeptidase, which has a wide specificity towards artificial substrates, was purified to homogeneity, as judged by SDS-PAGE, from the supernatant fluid of cells grown in continuous culture in a tryptone/glucose/thiamine medium. The enzyme has a molecular mass of 61 kDa, a pl of 6.3, a pH optimum close to 7.5 and showed a requirement for magnesium or calcium ions. The N-terminal sequence of the first 10 amino acids (Asp-Val-Asn-Met-Leu-Trp-Tyr-Val-x-Arg...) showed no similarity to any published sequence. This enzyme in its cell-bound or extracellular form may be important in the nutrition and pathogenesis of C. gingivalis in the human oral cavity.

Diversity of oral asaccharolytic Eubacterium species in periodontitis--identification of novel phylotypes representing uncultivated taxa.

Oral asaccharolytic Eubacterium species are associated with periodontal disease and other oral infections. The aim of this study was to use a culture-independent molecular technique to determine the diversity of asaccharolytic Eubacterium species in subgingival plaque in periodontitis. An oligonucleotide PCR primer designed to amplify 16S rRNA genes of the oral asaccharolytic Eubacterium branch of the phylogenetic tree was constructed. This primer was used together with a universal primer in PCRs to amplify gene sequences directly from a single subgingival plaque sample. Fifty PCR products were purified by cloning, fully sequenced and subjected to molecular phylogenetic analysis. The sequences were assigned to four groups within a single lineage of the low G + C gram-positive bacteria. Group I (58% of the cloned sequences) was assigned to a branch that included Eubacterium nodatum, and Group II (22%) to a branch including Eubacterium infirmum. Group III (8%) was distinct from but related to E. infirmum at the species level, and Group IV (12%) was another novel taxon more distantly related to E. infirmum and E. nodatum.

Growth and hydrolytic enzyme production of Capnocytophaga gingivalis on different protein substrates.

Capnocytophaga gingivalis was grown with proteins (albumin, collagen, mucin and hemoglobin) as carbon and energy sources in chemostat culture. The mu max (0.34 h-1) and biomass yield (0.96 g.l-1) were as high with hemoglobin (3 g.l-1) as with glucose (3 g.l-1) (20). Albumin, collagen and mucin also supported an increased mu max, or yield or both, in comparison with basal (tryptone/thiamine) medium. In steady-state, trypsin-like protease specific activity increased 3- to 5-fold in the presence of albumin, collagen and hemoglobin: whereas the greatest increase (21-fold) in alpha-glucoside activity was in the presence of mucin. There were significant, but less substantial changes in other hydrolytic enzymes (aminopeptidase, acid and alkaline phosphatases). The bulk of the detected hydrolytic activity (> 66%) was associated with the cells. The data indicate that C. gingivalis regulates its production of hydrolytic enzymes in response to environmental conditions.

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.

Capnocytophaga granulosa and Capnocytophaga haemolytica: novel species in subgingival plaque.

BACKGROUND: The oral cavity accommodates one of the most diverse microfloras in the human body. Knowledge of this microflora, and of the periodontal microflora in particular, proves crucial towards an understanding of the bacterial-host interactions which lead to the development of infectious inflammatory periodontal diseases. Capnocytophaga species have been implicated as putative periodontal pathogens. To date, only 3 members of this genus (C. gingivalis, C. ochracea and C. sputigena) have been isolated from subgingival plaque. AIM: This communication reports the isolation of 2 recently-speciated strains, namely C. granulosa and C. haemolytica, from subgingival plaque collected from adult periodontitis patients. MATERIAL AND METHODS: Subgingival plaque was collected from 29 patients with chronic adult periodontitis. Plaque samples were inoculated onto fastidious anaerobe agar and incubated anaerobically for 5 days. Routine identification of clinical isolates was performed by 16S rRNA PCR-RFLP analysis, using Cfo I as restriction enzyme and corroborated by 16S rRNA gene sequencing. RESULTS: 16 of 29 patients (55%) tested positive for either C. granulosa and or C. haemolytica. A total of 70 isolates (63 C. granulosa and 7 C. haemolytica) were cultivated from subgingival plaque. 15 (51%) patients tested positive for C. granulosa, and 3 (10%) patients tested positive for C. haemolytica. CONCLUSION: This is the 1st report which recounts the presence of C. granulosa and C. haemolytica in subgingival plaque. Further research is required to establish the relative proportions of these species subgingivally in health and disease.