The effectiveness of enzymic irrigation in removing a nutrient-stressed endodontic multispecies biofilm.

AIM: To establish a nutrient-stressed multispecies model biofilm and investigate the dynamics of biofilm killing and disruption by 1% trypsin and 1% proteinase K with or without ultrasonic activation. METHODOLOGY: Nutrient-stressed biofilms (Propionibacterium acnes, Staphylococcus epidermidis, Actinomyces radicidentis, Streptococcus mitis and Enterococcus faecalis OMGS 3202) were grown on hydroxyapatite discs and in prepared root canals of single-rooted teeth in modified fluid universal medium. The treatment groups included trypsin, proteinase K, 0.2% chlorhexidine gluconate and 1% sodium hypochlorite (NaOCl) (with and without ultrasonics). NaOCl and chlorhexidine were the positive controls and untreated group, and sterile saline was the negative control. The biofilms were investigated using confocal laser scanning microscopy (CLSM) with live/dead staining and quantitative microbial culture. RESULTS: Nutrient stress in the multispecies biofilm was apparent as the medium pH became alkaline, glucose was absent, and serum proteins were degraded in the supernatant. The CLSM showed the percentage reduction in viable bacteria at the biofilm surface level due to nutrient starvation. On the disc model, trypsin and proteinase K were effective in killing bacteria; their aerobic viable counts were significantly lower (P < 0.01) than the negative control and chlorhexidine. NaOCl was the most effective agent (P < 0.001). In the tooth model, when compared to saline, trypsin with ultrasonics caused significant killing both aerobically and anaerobically (P < 0.05). Chlorhexidine (1.46 ± 0.42), trypsin (3.56 ± 1.18) and proteinase K (4.2 ± 1.01) with ultrasonics were significantly effective (P < 0.05) in reducing the substratum coverage as compared to saline with ultrasonics (12% ± 4.9). CONCLUSION: Trypsin with ultrasonic activation has a biofilm killing and disrupting potential.

Dental caries from a molecular microbiological perspective.

Dental caries results from an imbalance of the metabolic activity in the dental biofilm. The microbial communities of teeth have traditionally been studied by standard cultural approaches. More recently, cloning and sequencing of the 16S rRNA gene have been used to characterize the microbial composition of the oral biofilm, but the methodological limitations of this approach have now been recognized. Next-generation high-throughput sequencing methods have the potential to reveal the composition and functioning of the biofilm by means of metagenomic and metatranscriptomic analyses. Currently available high-throughput sequencing approaches are reviewed and discussed in relation to studying the biofilm associated with dental caries. Important in understanding the dynamic processes in caries is the metabolic activity of the biofilm; metabolome analysis is a new tool that might enable us to assess such activity. As caries is a localized disease, it is essential that biofilm samples are taken from precisely determined tooth sites; pooling samples is not appropriate. This paper presents the case that culture-based studies are important, but that the fullest understanding of the role of the biofilm in the caries process will only come from an integrated approach determining biological function and metabolic output.

Fluoride-supplemented milk inhibits acid tolerance in root caries biofilms.

In this study we investigated the effect of fluoride on plaque acid tolerance. The test group consumed 200 ml of milk supplemented with 5 mg F/l as NaF once a day, the milk control group drank 200 ml of unsupplemented milk, and the no-milk control group did not consume milk in this manner. Plaque samples were taken at baseline and after 15 months. The proportion of acid-tolerant bacteria in plaque was estimated using LIVE/DEAD® BacLight™ staining after exposure to pH 3.5 for 2 h. The fluoride group showed a statistically significant decrease in plaque acid tolerance compared to baseline. This study shows that daily intake of fluoride in milk reduces plaque acid tolerance.

Multidisciplinary research agenda for novel antimicrobial agents for caries prevention and treatment.

Antimicrobial methods to augment fluoride-mediated caries inhibition are necessary. Several methods are described here, but none was considered likely to be as effective as fluoride usage. None had been tested in effective models to demonstrate their ability to act either additively or synergistically with fluoride-containing toothpastes. Dental caries is a biofilm-mediated disease: The composition of the biofilm associated with caries initiation and progression is diverse. Caries-associated taxa - including mutans streptococci, lactobacilli, bifidobacteria, and yeasts - may be useful surrogate markers for in vivo investigations. In vitro testing should progress from single-species planktonic cells to multi-species biofilms prior to essential testing in randomized control trials (RCTs). Modern high-throughput sequencing techniques need to be applied to the study of bacterial acquisition from birth and of the composition of the biofilm associated with the formation of white-spot lesions. The determination of the functions of the biofilm and the phenotype of the bacterial components may be determined by RNA-seq techniques, since they must be conserved between caries lesions and will include the ability to produce acids and survive and proliferate in acidic conditions. The application of such methods will significantly improve our understanding of the etiology and progression of dental caries.

Genetic diversity of Lactobacillus paracasei isolated from in situ human oral biofilms.

AIM: To determine the genetic diversity and possible origin of Lactobacillus paracasei found in the oral biofilm. METHODS AND RESULTS: Lactobacilli were isolated from a biofilm model, formed in situ prior to and during a period of exposure to 20% sucrose solution (28 days), using Rogosa Agar. The lactobacillus colonies were randomly selected (n = 222) and subcultured. The isolates were identified using pheS or rpoA gene sequence analysis. Lactobacilli identified as Lact. paracasei (n = 75) were subjected to multilocus sequencing typing (MLST) analysis by determining partial sequences of seven housekeeping genes fusA, ileS, lepA, leuS, pyrG, recA and recG. An increase recovery of lactobacilli after sucrose phase compared with nonsucrose period was observed (31 prior to and 191 following a sucrose exposure period). Seven subjects harboured Lact. paracasei and these represented 14 sequence types (ST). Comparison of the STs showed that unrelated subjects may harbour the same ST and that individuals harbour multiple STs. Three subjects harboured STs previously isolated from dairy products. CONCLUSION: The present data supports the hypothesis that oral lactobacilli may be of exogenous origin. SIGNIFICANCE AND IMPACT OF THE STUDY: The study allow us to gain insight into the genetic diversity of Lact. paracasei in oral biofilm.

Resistance to acidic environments of caries-associated bacteria: Bifidobacterium dentium and Bifidobacterium longum.

Oral Bifidobacteriaceae, Bifidobacterium dentium and Bifidobacterium longum, are known to be isolated together with mutans streptococci and lactobacilli from caries lesions, suggesting that these Bifidobacteriaceae are caries associated and acid resistant. This study aimed to investigate effects of acidification on B. dentium and B. longum, and to compare them with those on Streptococcus mutans, Streptococcus sanguinis and Lactobacillus paracasei. Effects of acidification, growth ability in a complex medium at a pH of 4.0-8.0, cell viability in 2-morpholinoethanesulfonic acid monohydrate (MES)-KOH buffer at pH 4.0, as well as stability of intracellular pH (pH(in)) at an extracellular pH of 3.5-8.0 estimated using a fluorescent dye, 5(6)-carboxyfluorescein diacetate N-succinimidyl ester in MES-KOH, 3-(N-morpholino)propanesulfonic acid-KOH or N,N-bis(2-hydroxyethyl)glycine-KOH buffer, were investigated. B. longum grew as well as Streptococcus strains over a wide pH range, whereas B. dentium grew best in the narrow pH range around neutral. The cell viability of B. dentium decreased significantly after 2 h of acidification at a pH of 4.0, but this was significantly less than that of the Streptococcus and Lactobacillus species, whereas B. longum maintained almost 100% viability. The pH(in) was close to the extracellular pH at pH of 5.5-7.5 in the Bifidobacterium and Streptococcus strains, while at a pH of <5.0, the pH(in) was higher than the extracellular pH in all the strains, but the pH(in) maintenance ability of Bifidobacterium strains was higher than that of the Streptococcus strains. The high survival rate and pH(in) maintenance ability of bifidobacteria comparable to that of S. mutans in the acidic environment may account for why bifidobacteria exist as stable species in acidic caries lesions together with mutans streptococci.

The effectiveness of passive ultrasonic irrigation on intraradicular Enterococcus faecalis biofilms in extracted single-rooted human teeth.

AIM: To compare the efficacy of passive ultrasonic irrigation with 1% sodium hypochlorite, with that of conventional syringe irrigation with 1% sodium hypochlorite, on intraradicular Enterococcus faecalis biofilms in extracted single-rooted human teeth. METHODOLOGY: Biofilms of E. faecalis (strain OMGS 3202) were grown on the prepared root canal walls of 48 standardized root halves which had been longitudinally sectioned. Following reapproximation, the roots were divided into four groups of twelve. The two experimental groups were treated with conventional syringe irrigation with 1% sodium hypochlorite solution (experimental group A) and passive ultrasonic irrigation with 1% sodium hypochlorite solution (experimental group B). Of the two control groups, the first was treated with conventional syringe irrigation with sterile saline solution (control group C), whilst the second control group (D) received no irrigation. The root halves were processed for scanning electron microscopy. Three images (x 700), coronal, middle and apical, were taken of the twelve root halves in each of the four groups, using a standardized protocol. The images were randomized and biofilm coverage assessed independently by three calibrated examiners, using a four-point scoring system. RESULTS: There were no significant differences in the scores for remaining biofilm coverage between group A (conventional syringe irrigation with 1% sodium hypochlorite) and group B (passive ultrasonic irrigation with 1% sodium hypochlorite) at the three observed levels. There was a significant difference between both experimental groups (groups A and B) and group C (conventional syringe irrigation with sterile saline solution) (P < 0.001) at all three observed levels. CONCLUSIONS: Both conventional syringe irrigation and passive ultrasonic irrigation with 1% sodium hypochlorite were effective at completely removing intraradicular E. faecalis biofilms. Conventional syringe irrigation with sterile saline solution was only partially effective at removing the biofilms.

Association between Bifidobacteriaceae and the clinical severity of root caries lesions.

BACKGROUND/AIMS: The isolation of members of the family Bifidobacteriaceae (bifids) from oral samples has been sporadic and a recent cloning study has suggested that they are not detectable in root caries lesions. METHODS: To better understand the presence of bifids in root caries we obtained clinical samples (15 of each) from sound exposed root surfaces, leathery remineralizing root lesions, and soft active root lesions. We investigated each for the presence of bifids using a mupirocin-containing selective medium and identified the isolates using 16S recombinant RNA sequencing. RESULTS: The proportion of bifids, as a percentage of the total anaerobic count, was significantly related to the clinical status of the sites sampled, being 7.88 +/- 1.93 in the infected dentine from soft lesions, 1.61 +/- 0.91 in leathery lesions, and 0.05 +/- 0.39 in plaque from sound exposed root surfaces. Bifids were isolated from all soft lesions, 13 of 15 leathery lesions, and five of the plaque samples. Bifidobacterium dentium was isolated from four of the plaque samples, from 13 samples from leathery lesions, and from 12 of the 15 samples of infected dentine from the soft active lesions. Parascardovia denticolens and Scardovia genomospecies C1 were each isolated from samples associated with all three clinical conditions whereas Scardovia inopicata and Bifidobacterium subtile were both isolated from the infected dentine of the leathery and soft lesions. Bifidobacterium breve was isolated from the infected dentine of soft root caries lesions. CONCLUSION: Bifids may be routinely isolated from root caries lesions using appropriate cultural methods.

The isolation of bifidobacteria from occlusal carious lesions in children and adults.

The aim of this study was to enumerate and identify bifidobacteria from occlusal carious lesions in permanent and deciduous teeth. Samples of infected dentine were obtained from 24 active occlusal lesions in deciduous teeth and from 15 occlusal lesions in permanent teeth. Plaque samples from sound occlusal surfaces of 12 caries-free adults and 12 children were also obtained. The bifidobacterial strains were isolated in mupirocin-containing selective media, Gram-stained and subcultured for identification. Total bacterial counts were determined using fastidious anaerobic agar, and isolates were identified using genus-specific PCR primers and were confirmed by 16S rRNA sequencing. Bifidobacteria were isolated from 13 of the 15 occlusal lesions in the adults and formed 5.09 +/- 2.11% of the total cultivable flora. In the children, bifidobacteria were isolated from 16 of the 24 occlusal lesions and formed 7.4 +/- 2.6% of the total flora. No bifidobacteria were isolated from the occlusal surfaces of caries-free adults or children. A total of 424 bifidobacteria were identified and these were Bifidobacteriumdentium, Parascardovia denticolens, Scardoviainopicata, Bifidobacterium longum, Scardovia genomosp. C1 and Bifidobacterium breve. B. dentium was present in 14 out of the 16 bifidobacteria-positive samples from the lesions on the deciduous teeth and in 7 out of the 13 positive lesions in adults (p = 0.04). The present data suggest that bifidobacteria may play a role in the progression of occlusal caries lesions in both children and adults.

Diversity of Veillonella spp. from sound and carious sites in children.

Detailed data on the distribution of Veillonella in caries-free and caries-active subjects are scarce. We hypothesized that the diversity of the genus would be lower in caries lesions than in plaque from caries-free individuals. The proportions of Veillonella were not significantly different in the two groups. All isolates (n = 1308) were genotyped by REP-PCR, and different genotypes (n = 170) were identified by 16S rRNA, dnaK, and rpoB sequencing. V. parvula, V. dispar, and V. atypica were in both groups, V. denticariosi only in caries lesions, and V. rogosae only from the caries-free individuals (p < 0.009). Lesions were more likely to harbor a single predominant species (p = 0.0018). The mean number of genotypes in the lesions was less than in the fissure (p < 0.001) or buccal (p = 0.011) sites. The Veillonella from caries-free sites were more diverse than those from caries lesions, and may be related to the acidic environment of caries lesions.

The predominant cultivable Veillonella spp. of the tongue of healthy adults identified using rpoB sequencing.

The predominant Veillonella spp. were isolated from the dorsum surface of the tongues of 11 healthy adults and identified to species level using rpoB sequencing because 16S ribosomal RNA sequence analysis does not reliably differentiate between all members of this genus. In all, 253 isolates were identified and the mean proportion (+/- SE) of Veillonella spp. per sample was 16.2 (+/- 3.6) with a range of 3.0% to 36.3% of the total anaerobic colony count. The predominant species were Veillonella atypica (10/11), Veillonella dispar (9/11) and Veillonella rogosae (8/11) because they were isolated from the majority of subjects. Veillonella parvula was isolated from only one subject while Veillonella dentocariosi and Veillonella montpelleriensis were not isolated from any subject.

Assessment of the ozone-mediated killing of bacteria in infected dentine associated with non-cavitated occlusal carious lesions.

The ability of ozone to kill micro-organisms associated with non-cavitated occlusal caries was investigated. The occlusal surfaces were treated with ozone (n = 53) or air (n = 49) for 40 s, and the underlying infected dentine was exposed. There was no significant difference between the number of bacteria recovered from the ozone-treated and the control sites (p > 0.1). Treatment of the exposed dentine with ozone resulted in a just significant (p = 0.044) reduction in bacterial counts. Ozone treatment of non-cavitated occlusal lesions for 40 s failed to significantly reduce the numbers of viable bacteria in infected dentine beneath the demineralized enamel.

The potential for sodium hexametaphosphate (SHMP) found in common children drinks to limit acid production in the oral biofilm.

OBJECTIVE: Sodium hexametaphosphate (SHMP) is a widely used industrial preservative commonly found in children's drinks. In this paper we examined the effect of SHMP incorporated into children's drinks on acid production by the oral biofilm by monitoring salivary concentrations of lactic acid. MATERIALS AND METHOD: Twelve healthy adult subjects with an average age 36 years (range 26-54 years) consumed 10 ml from four children's beverages (Coca Cola and three types of Sunny Delight supplemented with SHMP) and a standard solution of sucrose. Saliva was collected at intervals following exposure of the oral biofilm to the drinks and the clearance of carbohydrates and the appearance of lactate was measured using standard enzymatic techniques. RESULTS: All the carbohydrates derived from the drinks were cleared from saliva within 15 min of consumption. Comparison of two drinks [Sunny D Normal and Sunny C] with the same carbohydrate, but different SHMP concentrations suggested that SHMP in these beverages had no significant effect on acid production. CONCLUSIONS: In this clinical study the role of SHMP, incorporated in common beverages, did not inhibit acid production from carbohydrates.

Oral health of children undergoing allogeneic bone marrow transplantation.

The objective of this study was to investigate changes in the oral health of children undergoing allogeneic bone marrow transplantation. The study group comprised 23 children undergoing allogeneic bone marrow transplantation and their matched controls. The study group comprised 23 children undergoing allogeneic bone marrow transplantation and their matched controls. Measurements were taken of the mean decayed, missing and filled surfaces and the mean decayed, missing and filled teeth in both deciduous and permanent dentition at baseline, the mean bacterial plaque and gingival inflammation indices and mucositis at specific event-related times during the transplantation period, were measured. The number of decayed, missing and filled surfaces in deciduous teeth was significantly greater in the transplant children than the matched controls (P < 0.05) at baseline. There was a significant increase in both the mean bacterial plaque score for the deciduous teeth (P < 0.003) and the permanent teeth (P < 0.001) and the mean gingival inflammation score for the deciduous (P < 0.001) and the permanent teeth (P < 0.001), at 7 days post-transplantation. At 4 months post-transplantation the plaque and gingival inflammation score had returned to baseline levels. There were significantly increased mean bacterial plaque and gingival inflammation scores during the period of intense immunosuppression following allogeneic bone marrow transplantation.

The pattern of change in salivary immunoglobulins and antibodies to S. mitis and S. oralis in children undergoing bone marrow transplantation: use of an indirect method of assessment.

The objective of this study was to assess the pattern of change in salivary immunoglobulins and antibodies to S. mitis and S. oralis in 23 children following allogeneic bone marrow transplantation and their matched controls. To overcome the difficulty of obtaining a sufficient quantity of whole saliva from very young, sick children saliva was collected in a 5-ml oral rinse of sterile normal saline. It was not possible to measure the volume of whole saliva in each rinse and the concentration of the salivary immunoglobulins and bacterial antibodies were estimated from 1 ml of oral rinse. Despite these shortcomings a pattern of change in the mean concentrations of total salivary IgA, secretory IgA, antibodies to S. mitis and S. oralis and total IgG at specific event- related times during the transplantation period has been demonstrated. There was a significant increase in the concentration of salivary IgG 7 days post-transplantation, followed by significant decreases in total salivary IgA, secretory IgA and antibodies to S. mitis after recovery of the peripheral neutrophil count above 0.5 x 10(9). The concentrations of total IgA and antibodies to S. oralis was significantly greater in the transplant group 119 days post-transplantation.

Synergistic degradation of bovine serum albumin by mutans streptococci and other dental plaque bacteria.

Mutans streptococci (Streptococcus mutans and Streptococcus sobrinus) exhibited low levels of proteolytic activity against the model protein substrate, FITC-labelled bovine serum albumin, when incubated alone. Inclusion of other members of the dental plaque microflora in the assay usually resulted in marked increases in the degree of proteolysis and a high level of synergy. Interactions between mutans streptococci and either Streptococcus oralis or Fusobacterium nucleatum gave rise to the greatest degree of synergistic proteolytic degradation.

Proteolytic activity of oral streptococci.

Streptococcus mutans and Streptococcus sobrinus were the least proteolytic of 8 species of oral streptococci while Streptococcus oralis and Streptococcus sanguis were the most proteolytic. Degradation of FITC-BSA was significantly correlated with the hydrolysis of synthetic endopeptidase substrates. As S. oralis strains proliferate in dental plaque in the absence of dietary food their success, in vivo, might be due partially to their greater proteolytic activity compared to other oral streptococci.

Protein expression by planktonic and biofilm cells of Streptococcus mutans.

Streptococcus mutans, a major causal agent of dental caries, functions in nature as a component of a biofilm on teeth (dental plaque) and yet very little information is available on the physiology of the organism in such surface-associated communities. As a consequence, we undertook to examine the synthesis of proteins by planktonic and biofilm cells growing in a biofilm chemostat at pH 7.5 at a dilution rate of 0.1 h(-1) (mean generation time=7 h). Cells were incubated with (14)C-labelled amino acids, the proteins extracted and separated by two-dimensional electrophoresis followed by autoradiography and computer-assisted image analysis. Of 694 proteins analysed, 57 proteins were enhanced 1.3-fold or greater in biofilm cells compared to planktonic cells with 13 only expressed in sessile cells. Diminished protein expression was observed with 78 proteins, nine of which were not expressed in biofilm cells. The identification of enhanced and diminished proteins by mass spectrometry and computer-assisted protein sequence analysis revealed that, in general, glycolytic enzymes involved in acid formation were repressed in biofilm cells, while biosynthetic processes were enhanced. The results show that biofilm cells possess novel proteins, of as yet unknown function, that are not present in planktonic cells.

Effect of acid shock on protein expression by biofilm cells of Streptococcus mutans.

Streptococcus mutans is a component of the dental plaque biofilm and a major causal agent of dental caries. Log-phase cells of the organism are known to induce an acid tolerance response (ATR) at sub-lethal pH values ( approximately 5.5) that enhances survival at lower pH values such as those encountered in caries lesions. In this study, we have employed a rod biofilm chemostat system to demonstrate that, while planktonic cells induced a strong ATR at pH 5.5, biofilm cells were inherently more acid resistant than such cells in spite of a negligible induction of an ATR. Since these results suggested that surface growth itself triggered an ATR in biofilm cells, we were interested in comparing the effects of a pH change from 7.5 to 5.5 on protein synthesis by the two cell types. For this, cells were pulse labeled with [(14)C]-amino acids following the pH change to pH 5.5, the proteins extracted and separated by two-dimensional (2D) electrophoresis followed by autoradiography and computer-assisted image analysis. A comparison between the cells incubated at pH 5.5 and the control biofilm cells revealed 23 novel proteins that were absent in the control cells, and 126 proteins with an altered relative rate of synthesis. While the number of changes in protein expression in the biofilm cells was within the same range as for planktonic cells, the magnitude of their change was significantly less in biofilm cells, supporting the observation that acidification of biofilm cells induced a negligible ATR. Mass spectrometry and computer-assisted protein sequence analysis revealed that ATR induction of the planktonic cells resulted in the downregulation of glycolytic enzymes presumably to limit cellular damage by the acidification of the external environment. On the other hand, the glycolytic enzymes in control biofilm cells were significantly less downregulated and key enzymes, such as lactate dehydrogenase were upregulated during pH 5.5 incubation, suggesting that the enhanced acid resistance of biofilm cells is associated with the maintenance of pH homeostasis by H+ extrusion via membrane ATPase and increased lactate efflux.

Production of specific glycosidase activities by Streptococcus intermedius strain UNS35 grown in the presence of mucin.

An isolate of Streptococcus intermedius from a brain abscess showed neuraminidase (sialidase), beta-D-galactosidase, N-acetyl-beta-D-glucosaminidase and N-acetyl-beta-D-galactosaminidase activities. The optimal pH values of these enzymes were 5.5-6.0, 5.5-6.0, 5.0-5.5 and 5.0-5.5, respectively. The km of the enzymes varied according to whether the type of substrate was chromogenic or fluorogenic; sialidase was most active at the lowest substrate concentrations, with a km of 0.01 mM. In semi-defined medium, with porcine gastric mucin--a model glycoprotein--as the sole source of fermentable carbohydrate, levels of the glycosidases were significantly increased. Addition of glucose to the mucin-containing medium, or growth of cells in media supplemented with glucose alone, repressed glycosidic activities and the majority of these were cell-associated. S. intermedius cells from cultures grown with mucin were able, simultaneously, to transport via sugar:phosphoenolpyruvate phosphotransferase (PTS) systems, monosaccharides which are constituents of carbohydrate side chains of glycoproteins. These cells also possessed significant levels of neuraminate-pyruvate lyase, involved in the intracellular catabolism of neuraminic acid; this was absent from cells grown with glucose. These mechanisms, collectively, may facilitate the persistence and growth of S. intermedius in vivo.