The oral microbiota is a reservoir for antimicrobial resistance: resistome and phenotypic resistance characteristics of oral biofilm in health, caries, and periodontitis.

BACKGROUND: Antimicrobial resistance (AMR) is an ever-growing threat to modern medicine and, according to the latest reports, it causes nearly twice as many deaths globally as AIDS or malaria. Elucidating reservoirs and dissemination routes of antimicrobial resistance genes (ARGs) are essential in fighting AMR. Human commensals represent an important reservoir, which is underexplored for the oral microbiota. Here, we set out to investigate the resistome and phenotypic resistance of oral biofilm microbiota from 179 orally healthy (H), caries active (C), and periodontally diseased (P) individuals (TRN: DRKS00013119, Registration date: 22.10.2022). The samples were analysed using shotgun metagenomic sequencing combined, for the first time, with culture technique. A selection of 997 isolates was tested for resistance to relevant antibiotics. RESULTS: The shotgun metagenomics sequencing resulted in 2,069,295,923 reads classified into 4856 species-level OTUs. PERMANOVA analysis of beta-diversity revealed significant differences between the groups regarding their microbiota composition and their ARG profile. The samples were clustered into three ecotypes based on their microbial composition. The bacterial composition of H and C samples greatly overlapped and was based on ecotypes 1 and 2 whereas ecotype 3 was only detected in periodontitis. We found 64 ARGs conveying resistance to 36 antibiotics, particularly to tetracycline, macrolide-lincosamide-streptogramin, and beta-lactam antibiotics, and a correspondingly high prevalence of phenotypic resistance. Based on the microbiota composition, these ARGs cluster in different resistotypes, and a higher prevalence is found in healthy and caries active than in periodontally diseased individuals. There was a significant association between the resistotypes and the ecotypes. Although numerous associations were found between specific antibiotic resistance and bacterial taxa, only a few taxa showed matching associations with both genotypic and phenotypic analyses. CONCLUSIONS: Our findings show the importance of the oral microbiota from different niches within the oral cavity as a reservoir for antibiotic resistance. Additionally, the present study showed the need for using more than one method to reveal antibiotic resistance within the total oral biofilm, as a clear mismatch between the shotgun metagenomics method and the phenotypic resistance characterization was shown.

Listerine® Products: An Update on the Efficacy and Safety.

UNLABELLED: In the 19th century, the mouthwash Listerine® was formulated from four essential oils. Later, the oils were replaced by their marker substances. To keep them in solution, 24-27% ethanol was added as a vehicle. This is an update of our previous review on the efficacy and safety of Listerine®. METHOD: PubMed was searched for clinical studies on the therapeutic benefits and safety of Listerine® from the end of 2011 to the end of October 2015. RESULTS: Sixteen studies were found and extracted. Three of the four 6-month studies were of sound confirmatory design. Two of these investigated Listerine® and one Listerine Zero®. The evidence of effectiveness for Listerine®, based on the bulk of three confirmatory studies and numerous exploratory studies carried out so far, is strong, but only moderate for Listerine® Zero and poor for Listerine® Cool Blue. In the three safety studies identified, we found methodological flaws that biased the results. CONCLUSIONS: Evidence is accumulating that Listerine® is effective in improving oral health, but the absence of systematic toxicological studies means that an accurate safety assessment cannot be made.

Prevalence of Candida albicans and Candida dubliniensis in caries-free and caries-active children in relation to the oral microbiota-a clinical study.

OBJECTIVES: The correlation between caries and the oral prevalence of Candida spp. in children is contradictory in literature. Thereby, authors focused on Candida albicans as the most isolated Candida species from the oral cavity. Therefore, the aim of the present study was to compare caries-free and caries-bearing children regarding their oral carriage of Candida spp. MATERIAL AND METHODS: Twenty-six caries-free (CF group) and 26 caries-active children (CA group) were included into this study. Three different types of specimens were assessed, saliva and plaque, and in the case of caries, infected dentine samples were microbiologically analyzed for aerobic and anaerobic microorganisms and their counts. Special attention was given to the differentiation between C. albicans and Candida dubliniensis. Additionally, different biochemical tests, VITEK 2 (VITEK®2, bioMérieux, Marcy-l'Etoile, France) and 16S and 18S ribosomal DNA (rDNA) sequencing, were applied for identification. RESULTS: The detection of C. albicans did not differ between the CF and CA groups. C. dubliniensis was never detected in any specimen of the CF group, but occurred in one quarter of the CA group (27 % in plaque, 23 % in saliva), thus leading to a statistically significant difference between the two groups (p < 0.05). In six of these cases, C. dubliniensis was detected concomitantly in saliva and plaque and once only in plaque. CA group harbored statistically more Streptococcus mutans than the control group revealing a correlation between S. mutans and C. dubliniensis regarding the caries group. CONCLUSIONS: This is the first study reporting a frequent detection of C. dubliniensis in caries-active children, which could have been underestimated so far due to difficulties in differentiation between this yeast species and C. albicans. CLINICAL RELEVANCE: Microbiological diagnostic-especially of oral Candida species-is an important determinant for identifying etiological factors of dental caries in children.

An oral health optimized diet can reduce gingival and periodontal inflammation in humans - a randomized controlled pilot study.

BACKGROUND: The aim of this pilot study was to investigate the effects of four weeks of an oral health optimized diet on periodontal clinical parameters in a randomized controlled trial. METHODS: The experimental group (n = 10) had to change to a diet low in carbohydrates, rich in Omega-3 fatty acids, and rich in vitamins C and D, antioxidants and fiber for four weeks. Participants of the control group (n = 5) did not change their dietary behavior. Plaque index, gingival bleeding, probing depths, and bleeding upon probing were assessed by a dentist with a pressure-sensitive periodontal probe. Measurements were performed after one and two weeks without a dietary change (baseline), followed by a two week transitional period, and finally performed weekly for four weeks. RESULTS: Despite constant plaque values in both groups, all inflammatory parameters decreased in the experimental group to approximately half that of the baseline values (GI: 1.10 ± 0.51 to 0.54 ± 0.30; BOP: 53.57 to 24.17 %; PISA: 638 mm(2) to 284 mm(2)). This reduction was significantly different compared to that of the control group. CONCLUSION: A diet low in carbohydrates, rich in Omega-3 fatty acids, rich in vitamins C and D, and rich in fibers can significantly reduce gingival and periodontal inflammation. TRIAL REGISTRATION: German Clinical Trials Register; https://www.germanctr.de (DRKS00006301). Registered on 2015-02-21.

A Preliminary Investigation on the Antimicrobial Activity of Listerine®, Its Components, and of Mixtures Thereof.

Listerine® is one of the most popular mouthwashes worldwide and claims to combat harmful bacteria. In the past century, its recipe was changed from an essential oil mouthwash to a five-component mixture (thymol, menthol, eucalyptol, and methyl salicylate dissolved in 27% ethanol). The aim of this study was to get preliminary information about the antimicrobial activities of individual Listerine® components and their mixtures. We tested the bacterial strains Streptococcus mutans, Enterococcus faecalis, and Eikenella corrodens and the yeast Candida albicans. The established minimum inhibitory concentration (MIC) assay and the minimum bactericidal/fungicidal concentration (MBC/MFC) assay were applied. None of the combinations of two phenols at the concentrations contained within Listerine® were associated with either an additive or synergistic effect. Thymol had lower MIC and MBC/MFC values than the other Listerine® components and Listerine® against E. corrodens and C. albicans. The mixtures consisting of eucalyptol, methyl salicylate, and thymol were the most effective against S. mutans and E. faecalis and more effective than Listerine®. Our results demonstrate that the phenols and their concentrations as contained within Listerine® could be further optimized in terms of selecting those which increase their general effectiveness, at concentrations that do not induce harm.

Supplementary sampling of obturation materials enhances microbial analysis of endodontic treatment failures: a proof of principle study.

OBJECTIVES: Root canal treatment failures often correlate with persistent biomaterial-associated endodontic infections. The aim of the present study was to assess the impact of endodontic obturation material sampling from root canals with posttreatment apical periodontitis on improving standard study protocols. MATERIALS AND METHODS: Samples from previously filled root canals and their corresponding endodontic filling materials were obtained from five root-filled teeth with posttreatment periradicular lesions. After cultivation, the isolated microorganisms were quantified and biochemically identified. Moreover, clone libraries were constructed after the amplification of bacterial 16S ribosomal DNA (rDNA) from the same samples. DNA from selected clones was sequenced to identify microbial species. Transmission electron microscopy (TEM) aided visualization of the detected bacteria. RESULTS: Overall, 22 taxa of the phyla Firmicutes, Actinobacteria, and Bacteroidetes were detected in both obturation and root canal samples by culture-dependent and culture-independent methods. Root canal fillings sheltered 17 species (3.30-7.50 × 10(3) CFU/ml). Of these, nine were detected solely in the retrieved obturation materials. The reinfected root canals harbored 13 taxa (3.48-7.36 × 10(3) CFU/ml). Obligate and facultative anaerobic bacteria prevailed. The number of different species ranged from 1 to 5 within a single sample. Fungi were not detected. CONCLUSIONS: Bacteria can colonize both root canals and endodontic fillings in vivo. CLINICAL RELEVANCE: Integrating the sampling of obturation materials with standard root canal sample collection offers a clearer insight into the actual microbial flora of reinfected root canals and improves the study protocols of secondary/persistent endodontic infections.

Novel Broad-Spectrum Antimicrobial Photoinactivation of In Situ Oral Biofilms by Visible Light plus Water-Filtered Infrared A.

Antimicrobial photodynamic therapy (APDT) has gained increased attention as an alternative treatment approach in various medical fields. However, the effect of APDT using visible light plus water-filtered infrared A (VIS + wIRA) on oral biofilms remains unexplored. For this purpose, initial and mature oral biofilms were obtained in situ; six healthy subjects wore individual upper jaw acrylic devices with bovine enamel slabs attached to their proximal sites for 2 h or 3 days. The biofilms were incubated with 100 µg ml(-1) toluidine blue O (TB) or chlorin e6 (Ce6) and irradiated with VIS + wIRA with an energy density of 200 mW cm(-2) for 5 min. After cultivation, the CFU of half of the treated biofilm samples were quantified, whereas following live/dead staining, the other half of the samples were monitored by confocal laser scanning microscopy (CLSM). TB- and Ce6-mediated APDT yielded a significant decrease of up to 3.8 and 5.7 log10 CFU for initial and mature oral biofilms, respectively. Quantification of the stained photoinactivated microorganisms confirmed these results. Overall, CLSM revealed the diffusion of the tested photosensitizers into the deepest biofilm layers after exposure to APDT. In particular, Ce6-aided APDT presented elevated permeability and higher effectiveness in eradicating 89.62% of biofilm bacteria compared to TB-aided APDT (82.25%) after 3 days. In conclusion, antimicrobial photoinactivation using VIS + wIRA proved highly potent in eradicating oral biofilms. Since APDT excludes the development of microbial resistance, it could supplement the pharmaceutical treatment of periodontitis or peri-implantitis.

Influence of a mouthwash containing hydroxyapatite microclusters on bacterial adherence in situ.

OBJECTIVE: The aim of the present study was to investigate the efficacy of a new preparation in dental prophylaxis containing zinc-carbonate hydroxyapatite microclusters (Biorepair) for oral biofilm management. METHODS AND MATERIALS: Initial biofilm formation was carried out in situ with bovine enamel slabs fixed to individual upper jaw splints worn by six subjects. Rinses with the customary preparation as well as with subfractions (hydroxyapatite microclusters in saline solution; liquid phase without particles) were adopted for 1 min in situ after 1 min of pellicle formation, and the bacterial colonization was recorded after 6 h and 12 h, respectively. Rinses with chlorhexidine served as a reference. The adherent microorganisms were quantified and visualized using DAPI staining and live-dead staining (BacLight). Furthermore, the effects on Streptococcus mutans bacteria were tested in vitro (BacLight). RESULTS: Application of the customary preparation and of the separate components distinctly reduced the initial bacterial colonization of the enamel surface in situ as visualized and quantified with all techniques. After 12 h, 1.3 × 10(7) ± 2.0 × 10(7) bacteria/cm² were detected on unrinsed control samples with DAPI staining; 2.4 × 10(6) ± 3.3 × 10(6) after application of Biorepair (12 h after CHX-rinse; 1.3 × 10(5) ± 9.2 × 10(4)). Also, pure hydroxyapatite microclusters in saline solution (2.1 × 10(6) ± 3.0 × 10(6)) as well as the liquid phase without particles (5.1 × 10(5) ± 3.3 × 10(5)) reduced the amount of adherent bacteria. Furthermore, antimicrobial effects on S. mutans were observed in vitro. CONCLUSION: The preparation is an effective compound for biofilm management in the oral cavity due to antiadherent and antibacterial effects. CLINICAL RELEVANCE: The tested mouthrinse seems to be a reasonable amendment for dental prophylaxis.

Comparison of different live/dead stainings for detection and quantification of adherent microorganisms in the initial oral biofilm.

OBJECTIVES: The aim of the present study was to investigate different fluorescence-based, two-color viability assays for visualization and quantification of initial bacterial adherence and to establish reliable alternatives to the ethidium bromide staining procedure. MATERIALS AND METHODS: Bacterial colonization was attained in situ on bovine enamel slabs (n = 6 subjects). Five different live/dead assays were investigated (fluorescein diacetate (FDA)/propidium iodide (PI), Syto 9/PI (BacLight®), FDA/Sytox red, Calcein acetoxymethyl (AM)/Sytox red, and carboxyfluorescein diacetate (CFDA)/Sytox red). After 120 min of oral exposure, analysis was performed with an epifluorescence microscope. Validation was carried out, using the colony-forming units for quantification and the transmission electron microscopy for visualization after staining. RESULTS: The average number of bacteria amounted to 2.9 ± 0.8 × 10(4) cm(-2). Quantification with Syto 9/PI and Calcein AM/Sytox red yielded an almost equal distribution of cells (Syto 9/PI 45% viable, 55% avital; Calcein AM/Sytox red 52% viable, 48% avital). The live/dead ratio of CFDA/Sytox red and FDA/Sytox red was 3:2. An aberrant dispersal was recorded with FDA/PI (viable 34%, avital 66%). The TEM analysis indicated that all staining procedures affect the structural integrity of the bacterial cells considerably. CONCLUSION: The following live/dead assays are reliable techniques for differentiation of viable and avital adherent bacteria: BacLight, FDA/Sytox red, Calcein AM/Sytox red, and CFDA/Sytox red. These fluorescence-based techniques are applicable alternatives to toxic and instable conventional assays, such as the staining procedure based on ethidium bromide. CLINICAL RELEVANCE: Differentiation of viable and avital adherent bacteria offers the possibility for reliable evaluation of different mouth rinses, oral medication, and disinfections.

Microscope-based imaging platform for large-scale analysis of oral biofilms.

A microscopic method for noninvasively monitoring oral biofilms at the macroscale was developed to describe the spatial distribution of biofilms of different bacterial composition on bovine enamel surfaces (BES). For this purpose, oral biofilm was grown in situ on BES that were fixed at approximal sites of individual upper jaw acrylic devices worn by a volunteer for 3 or 5 days. Eubacteria, Streptococcus spp., and Fusobacterium nucleatum were stained using specific fluorescence in situ hybridization (FISH) probes. The resulting fluorescence signals were subsequently tested by confocal laser scanning microscopy (CLSM) and monitored by an automated wide-field microscope-based imaging platform (Scan∧R). Automated image processing and data analysis were conducted by microscope-associated software and followed by statistical evaluation of the results. The full segmentation of biofilm images revealed a random distribution of bacteria across the entire area of the enamel surfaces examined. Significant differences in the composition of the microflora were recorded across individual as well as between different enamel surfaces varying from sparsely colonized (47.26%) after 3 days to almost full surface coverage (84.45%) after 5 days. The enamel plates that were positioned at the back or in the middle of the oral cavity were found to be more suitable for the examination of biofilms up to 3 days old. In conclusion, automated microscopy combined with the use of FISH can enable the efficient visualization and meaningful quantification of bacterial composition over the entire sample surface. Due to the possibility of automation, Scan∧R overcomes the technical limitations of conventional CLSM.

Awareness of electronic crimes related to E-learning among students at the University of Jordan.

The spread of e-learning as an alternative to traditional or face-to-face education has faced many problems and challenges in general and ethical and legal challenges in particular. This study aims to measure students' awareness of the safe use of technology and its tools in e-learning that is consistent with ethical and legal standards. The study attempts to reveal the degree of awareness of students of the University of Jordan about electronic crimes related to e-learning and the legal procedures and penalties related to electronic crimes in e-learning. Quantitative research methods were used. A questionnaire was established and distributed to students enrolled in the following online courses: Ethics and Human Values, Communication Skills, and National Culture. Analysis of the data revealed that students had a high awareness about cybercrime due to the widespread use of the internet by students as it became an integral part of their daily lives. The degree of awareness of student about legal procedures and penalties related to electronic crimes in e-learning was medium. This indicates students' lack of awareness of the effectiveness of procedures and penalties for electronic crimes that can be applied in e-learning due to the rapid transition in the learning process at the University of Jordan from traditional learning to distance e-learning that was imposed during the Corona pandemic. Based on these findings, the study presented a set of recommendations that could be implemented to increase awareness and maximize the benefit of using e-learning.

Influence of the long-term use of oral hygiene products containing stannous ions on the salivary microbiome - a randomized controlled trial.

Oral hygiene products containing tin are suitable to prevent erosive tooth wear, yet effects on the oral microbiota are not known yet. Therefore, this study determined the salivary microbiome of 16 participants using products with stannous ions for three years (TG) compared with a control group (CG) to assess their influence on the microbiota. Participants were included in a randomized controlled clinical trial (RCT) with biannual visits. Illumina Miseq sequencing revealed as most abundant genera: Streptococcus (TG 14.3%; CG 13.0%), Veillonella (TG 11.3%; CG 10.9%), Prevotella (TG 7.0%; CG 9.8%), Haemophilus (TG 6.6%; CG 7.2%), Porphyromonas (TG 5.9%, CG 5.1%), Leptotrichia (TG 5.8%; CG 4.9%), Actinomyces (TG 4.0%; CG 4.6%) and Neisseria (TG 5.4%; CG 4.2%). Beta-Diversity was not significantly different between groups at both time points, although significant differences between groups were found for certain taxa after three years. The genus Prevotella was found in higher abundance in CG whereas Neisseria and Granulicatella, health-associated taxa, were found more abundantly in TG. Salivary microbiota after three years reflected a composition associated with oral health, hence continual use as a preventive measure for dental erosion can be considered safe and benefitting oral health for patients with a high risk of erosion.

Maintaining blood-brain barrier integrity: pericytes perform better than astrocytes during prolonged oxygen deprivation.

The blood-brain barrier (BBB), consisting of specialized endothelial cells surrounded by astrocytes and pericytes, plays a crucial role in brain homeostasis. Many cerebrovascular diseases are associated with BBB breakdown and oxygen (O(2)) deprivation constitutes a critical factor that onsets its disruption. We investigated the impact of astrocytes and pericytes on brain endothelial cell permeability and survival during different degrees of O(2) deprivation. Prolonged exposure to 1% O(2) caused barrier breakdown and exposure to 0.1% O(2) dramatically accelerated disruption and induced cell death, mediated at least in part via caspase-3 activation. Reoxygenation allowed only cells exposed to 1% O(2) to re-establish barrier function. Notably co-culture with astrocytes and pericytes substantially enhanced barrier function under normoxic conditions, and produced differential responses during O(2) deprivation. At 1% O(2) astrocytes partially maintained barrier integrity whereas pericytes accelerated its disruption in the short-term, having positive effects only after prolonged exposure. Unexpectedly, at 0.1% O(2) pericytes were more effective than astrocytes in preserving barrier function although the protection afforded by both cells involved inhibition of caspase-3 pathways. Furthermore, cell-specific regulation of auto- and paracrine VEGF signaling pathways were also in part responsible for the differential modulation of barrier function. Our data suggests that cellular cross-talk within the neurovascular unit is crucial for preservation of barrier integrity and that pericytes, not astrocytes, play a significant role during severe and prolonged O(2) deprivation.

Initial Bacterial Adhesion Properties of Anodically Oxidized Ti6Al4V.

This paper reports about the initial interaction of bacteria with anodically oxidized Ti6Al4V for the use as dental implant abutment surfaces. Ti6Al4V samples are anodically oxidized in hydrofluoric acid using different voltages. The resulting nanotopographies are characterized by atomic force microscopy, scanning electron microscopy and contact angle measurements. The topographies reach from micro-porous structures with small nanoporosities on top to fully hexagonally aligned nanotubes. For initial bacterial adhesion tests, Escherichia coli and Staphylococcus aureus are used. Samples are incubated for 2 h and afterwards non-adherent cells are washed off. The results of live/dead staining and cell counts are presented. Gram-negative and Gram-positive strains show different behavior in respect to total number of initially adherent cells on different micro/nanotopographies. The observed reduction of adhered microorganisms is mainly based on underlying microporous topographies.

Comparative in vitro study of the proliferation and growth of human osteoblast-like cells on various biomaterials.

In vitro studies about the growth behavior of osteoblasts onto biomaterials is a basic knowledge and a screening method for the development and application of scaffolds in vivo. In this in vitro study human osteoblast-like (HOB) cells were cultured on seven different biomaterials used in dental and craniomaxillofacial surgery, respectively. The tested biomaterials were synthetic biodegradable (MacroPore, Ethisorb, PDS, Beriplast P) and nonbiodegradable polymers (Palacos) as well as calcium phosphate cement (BoneSource) and titanium. The cell proliferation and cell colonization were analyzed by scanning electron microscopy and EZ4U-test. Statistical analysis were performed. HOB-like cells cultivated on Ethisorb showed the highest proliferation rate. The proliferation rate was statistically significant compared with Palacos, MacroPore, and BoneSource. Whereas, Beriplast, PDS, and titanium yielded lower proliferation rates. However, there was no statistically significant difference compared with Palacos, MacroPore, and BoneSource. SEM analysis showed no significant difference in individual cell features and cell colonization. But an infiltration and a growth of HOB-like cells throughout the porous structure of Ethisorb, which is formed by crossing fibers, is a striking different feature (macrotopography). This feature can explain the highest proliferation rate of Ethisorb. The results showed that HOB-like cells appear to be sensitive to substrate composition and topography. Moreover, the basis for further studies with such biomaterial/osteoblast constructs in vivo are provided. Further focusing points are developing techniques to fabricate three-dimensional porous biomaterial/cell constructs, studying the tissue reaction and the bone regeneration of such constructs compared with the use of autologous bone.

Comparative in vitro study of the cell proliferation of ovine and human osteoblast-like cells on conventionally and rapid prototyping produced scaffolds tailored for application as potential bone replacement material.

Reconstruction of bone defects in the field of craniomaxillofacial surgery is a relevant problem. In regenerative medicine, autologous bone is not available sufficiently. The full replacement of autologous bone grafts is required. A promising research field is the bone engineering. Especially the application of rapid prototyping (RP) enables new perspectives concerning the scaffold design. The aim of the study was to compare scaffolds produced by RP-technology (native and plasma-coated PLGA-scaffolds) with conventionally produced scaffolds (agar plates with hydroxyapatite and hyaluronic acid coated agar plates with hydroxyapatite) relating to proliferation, adhesion, and morphology of osteoblasts to get knowledge about the application potential of such 3D-manufactured matrices for bone engineering. TissueFoil E served as reference. To compare the scaffolds, 12 ovine and 12 human osteoblast-like cell cultures of the skull were used. Results were obtained by EZ4U, scanning electron microscopy, and light microscopy. The highest cell proliferation rate of human osteoblast-like cells was measured on TissueFoil E followed by plasma-coated PLGA-scaffolds and uncoated PLGA-scaffolds, whereas of ovine osteoblast-like cells on plasma-coated PLGA-scaffolds followed by TissueFoil E and uncoated PLGA-scaffolds. Human and ovine osteoblast-like cells on coated and uncoated agar plates had significant lower proliferation rates compared with TissueFoil E and PLGA-scaffolds. These results showed the potential of RP in the field of bone engineering. Mechanical properties of such scaffolds and in vivo studies should be investigated to examine if the scaffolds hold up the pressure it will undergo long enough to allow regrowth of bone and to examine the revascularization.

Fluorescence microscopic visualization and quantification of initial bacterial colonization on enamel in situ.

OBJECTIVE: The acquired salivary pellicle has been defined as proteinaceous film free of bacteria. However, due to the large numbers of microorganisms existent in the oral fluids, it is conceivable that adherent bacteria are already present in the initial pellicle. The aim of this in situ study was to visualize and to quantify these bacteria. DESIGN: Initial biofilm formation was performed on bovine enamel slabs mounted buccally on individual splints and carried in situ by six subjects for 3, 30 and 120 min, respectively. After intraoral exposure, the slabs were rinsed with saline solution and the adherent bacteria were investigated with the following fluorescence microscopic methods: staining with 4',6-diamidino-2-phenylindole (DAPI), staining of vital and nonvital bacteria with fluoresceinediacetate and ethidiumbromide (live/dead staining) and fluorescence in situ hybridization (FISH) of eubacteria and streptococci, respectively. In addition, determination of colony forming units after ultrasonically induced detachment of bacteria was performed. RESULTS: With all the methods, bacteria were detected in the initial in situ biofilm irrespective of the formation time. The numbers of bacteria revealed high intraindividual and interindividual variability and the microorganisms were distributed randomly in small aggregates. The results of the epifluorescence microscopic techniques corresponded well. The mean number of adherent bacteria detected was in the range of 10-20x10(4)cm(-2). CONCLUSION: Already after 3 min, adherent bacteria are present in the initial pellicle. For the first time, DAPI-staining as well as FISH have proven success for visualization of initial intraoral colonization of enamel specimens.

Transport of quercetin di-sodium salt in the human intestinal epithelial Caco-2 cell monolayer 139.

Quercetin di-sodium salt (QDS), a water-soluble derivative of quercetin (Q), is a potent free radical scavenger. The aim of this study was to examine the in vitro intestinal transport of QDS compared to that of Q using the Caco-2 human intestinal epithelial cell line. The apical (A) to basolateral (B) transport of QDS was found to be higher than the B to A transport of this compound. This polarized transport involved the presence of a carrier protein system. The involvement of the sodium/glucose transporter-1 (SGLT-1) was shown by using phloridzin, a selective inhibitor of this conveyor system. However, the transport of Q was not affected by this inhibitor. Moreover, the influx of QDS was pH-sensitive and decreased at pH 5.5 compared with that observed at pH 7.4 and 6.5. The permeability of QDS was 10-fold higher than that of Q. This could be explained by the involvement of SLGT-1 and the absence of an active efflux pump in the absorption of QDS in comparison with Q. This finding was supported by comparing the solubility of Q with that of QDS. This study indicates that both the higher solubility of QDS and its dependence on the SGLT-1 transport system resulted in more efficient permeability compared to Q.

Reduced kidney function and anemia as risk factors for mortality in patients with left ventricular dysfunction.

OBJECTIVES: We sought to evaluate the relationship between the level of kidney function, level of hematocrit and their interaction on all-cause mortality in patients with left ventricular (LV) dysfunction. BACKGROUND: Anemia and reduced kidney function occur frequently in patients with heart failure. The level of hematocrit and its relationship with renal function have not been evaluated as risk factors for mortality in patients with LV dysfunction. METHODS: We retrospectively examined the Studies Of LV Dysfunction (SOLVD) database. Glomerular filtration rate (GFR) was predicted using a recently validated formula. Kaplan-Meier survival analyses were used to compare survival times between groups stratified by level of kidney function (predicted GFR) and hematocrit. Cox proportional-hazards regression was used to explore the relationship of survival time to level of kidney function, hematocrit and their interaction. RESULTS: Lower GFR and hematocrit were associated with a higher prevalence of traditional cardiovascular risk factors. In univariate analysis, reduced kidney function and lower hematocrit, in men and in women, were risk factors for all-cause mortality (p < 0.001 for both). After adjustment for other factors significant in univariate analysis, a 10 ml/min/1.73 m(2) lower GFR and a 1% lower hematocrit were associated with a 1.064 (95% CI: 1.033, 1.096) and 1.027 (95% CI: 1.015, 1.038) higher risk for mortality, respectively. At lower GFR and lower hematocrit, the risk was higher (p = 0.022 for the interaction) than that predicted by both factors independently. CONCLUSIONS: Decreased kidney function and anemia are risk factors for all-cause mortality in patients with LV dysfunction, especially when both are present. These relationships need to be confirmed in additional studies.