Antimicrobial efficiency and cytocompatibility of different decontamination methods on titanium and zirconium surfaces.

OBJECTIVES: The purpose of this study was to investigate the efficiency of different implant-decontamination methods regarding biofilm modification and potential cytotoxic effects. Therefore, the amount of biofilm reduction, cytocompatibility, and elementary surface alterations were evaluated after decontamination of titanium and zirconium surfaces. MATERIAL AND METHODS: Titanium and zirconium disks were contaminated with a newly developed high-adherence biofilm consisting of six microbial species. Decontaminations were performed using titanium curette, stainless steel ultrasonic scaler (US), glycine (GPAP) and erythritol (EPAP) powder air-polishing, Er:YAG laser, 1% chlorhexidine (CHX), 10% povidone-iodine (PVI), 14% doxycycline (doxy), and 0.95% NaOCl solution. Microbiologic analysis was done using real-time qPCR. For assessment of cytocompatibility, a multiplex assay for the detection of cytotoxicity, viability, and apoptosis on human gingival fibroblasts was performed. X-ray photoelectron spectroscopy (XPS) was used to evaluate chemical alterations on implant surfaces. RESULTS: Compared with untreated control disks, only GPAP, EPAP, US, and Er:YAG laser significantly reduced rRNA counts (activity) on titanium and zirconium (p < .01), whereas NaOCl decreased rRNA count on titanium (p < .01). Genome count (bacterial presence) was significantly reduced by GPAP, EPAP, and US on zirconium only (p < .05). X-ray photoelectron spectroscopy analyses revealed relevant re-exposure of implant surface elements after GPAP, EPAP, and US treatment on both materials, however, not after Er:YAG laser application. Cytocompatibility was impaired by CHX, PVI, doxy, and NaOCl. CHX and PVI resulted in the lowest viability and doxy in the highest apoptosis. CONCLUSIONS: Within the limits of this in vitro study, air-polishing methods and ultrasonic device resulted in effective biofilm inactivation with surface re-exposure and favorable cytocompatibility on titanium and zirconium. Chemical agents, when applied on implant surfaces, may cause potential cytotoxic effects.

Evaluation of hydrocortisone as a strain-dependent growth-regulator of Porphyromonasgingivalis.

OBJECTIVE: Porphyromonas gingivalis is an oral key pathogen and known to be very diverse in geno- and phenotypes. It is a fastidious bacterium with low O2-tolerance and 3-7 days of incubation are necessary. With growing interest in the field of microbial endocrinology we explored the potential growth-stimulating effect of hydrocortisone (HC, synonym cortisol) on P. gingivalis cultures. MATERIAL AND METHODS: Six different P. gingivalis strains were pre-incubated in supplemented Brain-Heart-Infusion broth under appropriate conditions for 24 h, diluted and transferred into microplates. A newly developed and semi-automated spectrophotometric measurement in triplicate, applying a SpectraMax i3x microplate reader at an optical density of 600 nm, was conducted to test growth differences between test group (exposed to a supplement of either 1.25, 2.5, 5, 10, or 20 µg/ml of hydrocortisone) and control group over 48 h of anaerobic incubation (O2 ≤ 1%). Furthermore, strains were also incubated on HC-supplemented blood agar to test for a possible growth-stimulating effect on solid media. RESULTS: HC significantly stimulated the lag-phase growth of four out of six P. gingivalis strains. Our data suggest a concentration-dependent growth stimulatory effect of HC between 2.5 and 5 µg/ml, while below 1.25 µg/ml and above 10 µg/ml HC either did not stimulate or inhibited growth. CONCLUSIONS: HC could reduce the incubation time when isolating P. gingivalis from clinical samples and could boost low biomass cultivations especially during their lag-phase. The growth-modulating effect might be via modulation of virulence factors/quorum sensing gene expression or by reactive oxygen species(ROS)-capturing during early stages of bacterial growth. Further experiments are necessary to explain the mechanism behind our observations.

Isoeugenol-functionalized nanogels inhibit peri-implantitis associated bacteria in vitro.

OBJECTIVES: Obligate and facultative anaerobic bacteria adhering to dental implants are a major cause for peri-implant inflammation, which, if left untreated, can lead to implant loss. Previously, our group developed a new route for the synthesis of isoeugenol-functionalized aqueous nanogels for implant coatings. METHODS: Here, the antimicrobial activity of several new nanogels differing in spacer length (n = 6, 9, 44), radius (60-200 nm), and amount of isoeugenol functional substance (1-20 mol%) was tested against the following peri-implantitis-associated species: Fusobacterium nucleatum, Porphyromonas gingivalis, Prevotella intermedia, Aggregatibacter actinomycetemcomitans, Escherichia coli, Actinomyces viscosus, Enterococcus faecalis, Staphylococcus aureus, Streptococcus oralis, S. parasanguinis, and the yeast Candida albicans. The minimal bactericidal concentration (MBC) and fungicidal concentration (MFC) were determined for each combination. In addition, transmission electron microscopy (TEM) and fluorescence microscopy after live-dead-staining (LD-S) were performed to visualize nanogel-microbe interactions. RESULTS: Two nanogels, NG9-3 and NG9-4 (colloids of 80-150 nm, with a spacer length of n = 9 and feeding between 5 and 10 mol% isoeugenol), had an inhibitory effect on all Gram-positive species and on P. gingivalis and P. intermedia with MBC ≥31.25 µg/ml. TEM and LD-S images showed that cellular adhesion and uptake of nanogels resulted in swelling, shedding, or even complete detachment of the cell wall and then to bursting (see graphical abstract). CONCLUSIONS: Functional nanogels can be used as building blocks in the design of bioactive coatings on implants to prevent infection and accelerate tissue regeneration, but the concentrations required are higher than for antibiotics.

The Effect of Solutions Containing Extracts of Vochysia tucanorum Mart., Myrcia bellaCambess., Matricaria chamomilla L. and Malva sylvestris L. on Cariogenic Bacterial Species and Enamel Caries Development.

This study evaluated the effect of experimental solutions containing plant extracts on bacterial species and enamel caries prevention. Microcosm biofilm was produced from human saliva mixed with McBain saliva (0.2% sucrose) on bovine enamel for 5 days (3 days under anaerobiosis and 2 days under aerobiosis) at 37°C. From the 2nd day, the following treatments were applied (1 × 60 s/day): Vochysia tucanorum (10 mg/mL); Myrcia bella (5 mg/mL); Matricaria chamomilla (80 mg/mL); Malva sylvestris, fluoride, and xylitol (Malvatricin Plus®); 0.12% chlorhexidine (CHX, PerioGard®); and PBS (negative control). The medium pH was measured. Quantitative polymerase chain reaction was performed for the detection of Streptococcus mutans and Lactobacillus spp. Enamel demineralization was measured by spectral-domain optical coherence tomography. The data were compared by means of the Kruskal-Wallis/Dunn, two-way ANOVA/Bonferroni, and ANOVA/Tukey tests (p < 0.05). The pH decreased after sucrose exposure; only CHX reestablished pH >5.5 by the last day. CHX also eliminated Lactobacillusspp., but the other treatments did not differ significantly from PBS. Malvatricin Plus® and CHX eliminated S. mutans, but the other treatments did not differ from PBS. Similar results were seen concerning the reduction of lesion depth and reflectivity. The experimental natural-extract solutions were ineffective against cariogenic bacteria and in preventing the development of enamel caries.

Analysis of relative bacterial activity and lactate dehydrogenase gene expression of caries-associated bacteria in a site-specific natural biofilm: an ex vivo study.

OBJECTIVES: Detecting bacterial activity is considered a promising approach to monitor shifts from symbiosis to dysbiosis in oral microbiome. The present study aimed at investigating both the relative bacterial activity and the lactate dehydrogenase (ldh) gene expression of caries-associated bacteria in a site-specific natural biofilm. MATERIAL AND METHODS: Sixty subjects (age, mean ± SE: 30.1 ± 1.4) were allocated to two groups: caries-free subjects (CF) or caries-active subjects (CA). CF presented one sound surface (CFS, n = 30). CA presented two donor sites: a cavitated caries lesion (CAC, n = 30) and a sound reference surface (CAS, n = 30). Real-time quantitative PCR (q-PCR) on species or genus level and total bacteria was performed targeting the 16S gene, the 16S rRNA, the ldh gene, and the ldh mRNA (increasing 16S ribosomal RNA copy numbers can function as an indicator of increased energy metabolism). As the 16S rRNA abundance represents the number of ribosomes, while the 16S gene abundance represents the number of genomes, the quotient of the relative abundances functions as a measure for the relative bacterial activity (%). RESULTS: Both lactobacilli and S. mutans showed the highest relative bacterial activity in CAC ((mean ± SE) 218 ± 60% and 61 ± 16%, respectively) and the lowest values for both sound reference surfaces (69 ± 48%; 8 ± 3%). Significant differences were found between CAC and CAS as well as between CAC and CFS for both lactobacilli and S. mutans (p < 0.05). The ldh gene expression of lactobacilli and S. mutans only showed moderate values in CAC (1.90E+03 ± 2.11E+03; 2.08E+04 ± 4.44E+04 transcripts/µl) and CFS (2.04E+03 ± 2.74E+03; 8.16E+03 ± 6.64E+03 transcripts/µl); consequently no significant differences were detected. CONCLUSION AND CLINICAL RELEVANCE: Caries-associated bacteria (lactobacilli and S. mutans) showed the highest relative bacterial activity in plaque of cavitated lesions, the lowest in sound surfaces, allowing the detection of a significant activity shift in health and disease for caries-active patients. However, no significant differences in ldh gene expression could be determined.

Early and mature biofilm on four different dental implant materials: An in vivo human study.

OBJECTIVES: The aim of this study was to examine the microbial composition of early (after 3 days, D3) and mature biofilms (after 31 days, D31) on materials typically used in implant/abutment buildups. Implant/abutment materials with different surface roughness values (Ra ) were compared to detect differences in the quantity and quality of bacterial composition. MATERIAL AND METHODS: Four different materials were investigated: rough implant surface (sand-blasted acid-etched titanium, Ti-p), implant collar (machined titanium, Ti-m), titanium abutment (Ti6Al4V), and zirconium dioxide abutment (ZrO2 ). Fourteen periodontally healthy subjects received mandibular acrylic devices with four disks (one for each material) facing the anterior lingual area. The total bacterial count was analyzed using RT-qPCR. Both presence and proliferation of 20 selected bacterial species were assessed with microarrays. RESULTS: The highest mean total cell counts (x108  ± standard deviation) were detected at D3 on ZrO2 (5.63 ± 4.83; Ra  = 0.74 µm), followed by Ti-p (4.53 ± 5.00; Ra  = 1.87), Ti-m (4.43 ± 9.38; Ra  = 0.18 µm), and Ti6Al4V (3.83 ± 3.13; Ra  = 0.16 µm). ZrO2 showed significantly higher total bacterial cell counts than Ti-p and Ti-m (p < .05) for both time intervals. The microarrays detected 16 (D3) and 17 (D31) bacterial species; those associated with healthy oral microbiotas, but also bacteria of the red complex (Tannerella forsythia, Treponema denticola), were found on all materials. CONCLUSIONS: Biofilms on ZrO2 harbored a higher total number of bacterial cells compared with those formed on titanium surfaces with much lower roughness values. Putative periodontopathogens were detected on all materials after both time intervals. Implant/abutment materials with a low surface roughness showed less biofilm accumulation.

Biofunctionalized zinc peroxide nanoparticles inhibit peri-implantitis associated anaerobes and Aggregatibacter actinomycetemcomitans pH-dependent.

For dental implants the accumulation of anaerobic bacteria is a main reason for peri-implant inflammation, which untreated can lead to implant loss. Oxygen releasing substances may act as antibacterial agents. In this study glucose-1-phosphate (Glc-1P) biofunctionalized zinc peroxide (ZnO2) nanoparticles of four different synthesis ratio (1-10:1) and sizes (4-5 nm) were tested against the anaerobes Fusobacterium nucleatum, Porphyromonas gingivalis, and Prevotella intermedia, as well as against Aggregatibacter actinomycetemcomitans, Enterococcus faecalis, Staphylococcus aureus, Lactobacillus paracasei, and the yeast Candida albicans. Nanoparticles stabilized with o-phosphorylethanolamine, bis[2-(methacryloyloxy)ethyl] phosphate, or dioctyl sulfosuccinate instead of glucose were used as controls. For every combination of test strain and nanoparticle both, the minimal inhibitory (MIC) and minimal microbicidal concentration (MBC or MFC) were determined under different pH conditions in microtiter plates. Furthermore, transmission electron (TEM) and fluorescence microscopy after live-dead-staining was performed on selected combinations of pathogen and nanoparticle in order to visualize the interactions. The ZnO2/Glc-1P nanoparticles had an inhibitory effect on gram-negative anaerobes and on A. actinomycetemcomitans with a pH-dependent MIC ≥25 µg/ml and MBC ≥50 µg/ml, while the gram-positive species tested and C. albicans were not inhibited. In TEM images, attachment of nanoparticle-chains to the bacterial outer membrane and subsequent penetration was found together with an intracellular oxygen release. For nanoparticles with other stabilizers than glucose an invasion was only seen in elongated, dividing cells, possibly because of the more porous cell wall in the parting layer. Decorating ZnO2 by glucose-1-phosphate is a Trojan horse approach to permit their uptake in gram-negative oxygen-sensitive bacterial cells.

Evaluation of Restorative Materials Containing Preventive Additives in a Secondary Caries Model in vitro.

The incorporation of antibacterial agents into dental restorative materials is a promising strategy for secondary caries prevention. Previously, Carolacton affected Streptococcus mutans biofilm formation on composite materials in vitro. The present study evaluated secondary caries formation adjacent to Carolacton-containing composites and conventional restorative materials using an artificial biofilm model. Standardized cavities were prepared in bovine dentin-enamel samples (n = 175) and restored with various dental materials (Tetric EvoCeram [T], GrandioSo composite without [G] and with Carolacton [GC], Grandio Flow without [F] and with Carolacton [FC], GrandioSo containing sodium fluoride [GNaF], and Ketac Fil [K]). After artificial aging, S. mutans was grown on the samples for 7 days. The investigation of gap sizes and secondary caries formation was performed using confocal laser scanning microscopy and transversal microradiography. Median gap size in enamel was 9.4 µm (interquartile range 7.9-12.7). Compared to all other groups significant differences in gap sizes could be observed for Ketac Fil (p < 0.001; Mann-Whitney test). Only GrandioSo composite containing 30% sodium fluoride and Ketac Fil showed significantly smaller lesion areas in enamel (p < 0.001; Mann-Whitney test) than all other groups which was confirmed by the mineral loss data (p < 0.001; Mann-Whitney test). Based on the present in vitro results, it seems that Carolacton-containing composite in the current formulation within the shown simplified monoculture biofilm model is not able to prevent caries formation compared to fluoride-releasing restorative materials.

Correlation between relative bacterial activity and lactate dehydrogenase gene expression of co-cultures in vitro.

OBJECTIVES: The present study aims at correlating the relative bacterial activity with the H+ concentration and the ldh expression of caries-associated bacteria in co-cultures. MATERIALS AND METHODS: Well plates were prepared with BHI medium and cultures of Lactobacillus paracasei and Fusobacterium nucleatum. Bacterial growth at 37 °C was measured using a microplate-photometer before and after adding sucrose to the samples. Samples of co-cultures (n = 12) and single-species cultures (n = 3) were taken and pH was assessed. Real-time quantitative PCRs were applied targeting the 16S-gene, the 16S-rRNA, the ldh-gene, and the ldh-mRNA. RESULTS: For L. paracasei with sucrose, an increase in relative bacterial activity (62.8% ± 23.5% [mean, SE]) was observed, while F. nucleatum showed a clear decrease in relative bacterial activity (- 35.0% ± 9.6%). Simultaneously, the H+ concentration increased (1.15E-05 mol*l-1 ± 4.61E-07 mol*l-1). Consequently, a significant positive correlation was found between L. paracasei's relative bacterial activity and H+ concentration (Spearman rank correlation, r = 0.638; p = 0.002), while F. nucleatum exhibited a negative correlation (r = - 0.741; p ≤ 0.001). Furthermore L. paracasei with sucrose showed a moderate, but significant positive correlation between relative bacterial activity and ldh-expression (r = 0.307; p = 0.024). CONCLUSIONS AND CLINICAL RELEVANCE: The relative bacterial activity after sucrose pulse showed a significant correlation not only to the acid production (H+ concentration) but also to ldh expression of L. paracasei. However, further research is required to confirm these findings in a mature biofilm in vivo.

Challenges of next-generation sequencing targeting anaerobes.

Next-generation sequencing allows for investigating the composition of microbiomes that are associated with infection (clinical microbiology) or dysbiosis (microbial ecology). The most commonly applied short-read sequencing technologies are Illumina MiSeq/HiSeq and Ion Torrent PGM, however, other platforms that generate long-reads are under way and optimized. A pre-condition for representative results is an appropriate method for contamination-free collection, homogenization, storage of specimens and a subsequent efficient DNA extraction protocol. As some of the anaerobes such as Clostridia or anaerobe Archaea are robust while others of the same environment, such as spirochetes, possess a very thin cell wall, a chemico-mechanical lysing strategy is recommended but with some precautions to avoid DNA-sheering and overheating. For amplicon sequencing, the Silva-TestPrime online tool helps to find the optimal 16S directed primers for individual studies. For metagenome profiling, the classifier tool has to be selected with helpful decision trees available but a combination based on different strategies seems to be indispensable. Further development of both hard- and software is needed before microbiome results become free of a substantial technology-dependent bias.

Age-related changes in immune function (immune senescence) in caries and periodontal diseases: a systematic review.

AIM: To systematically review the evidence regarding immune senescence in the pathogenesis of periodontitis and dental caries. METHODS: A systematic search of electronic databases utilizing medical subject headings (MeSH terms) supplemented by screening of review articles and other relevant texts was undertaken. RESULTS: Seventy-three articles were included (43 for periodontitis, 30 for caries). Study results were found to be generally heterogeneous. Regarding periodontitis, human studies suggest evidence for altered neutrophil function and increased production of pro-inflammatory mediators (e.g. interleukin-1ß, interleukin-6 and prostaglandin E2 ) in older compared to younger subjects, and animal experiments suggest increased expression of genes that contribute to a pro-inflammatory state in older compared to younger animals. Regarding dental caries, research relating to changes in immune functioning and the impact of ageing is in its infancy. A small number of studies have reported components of innate and adaptive immunity that affect the composition of saliva and dental biofilms with possible impacts on caries progression. CONCLUSION: There is evidence that immune functioning related to periodontitis and (less investigated) dental caries alters with increasing age. In both conditions, age-associated mechanistic changes in immune functioning are complex and incompletely understood and it is not clear how these relate to disease susceptibility.

Dental caries and periodontal diseases in the ageing population: call to action to protect and enhance oral health and well-being as an essential component of healthy ageing - Consensus report of group 4 of the joint EFP/ORCA workshop on the boundaries between caries and periodontal diseases.

BACKGROUND: Over the last two decades, progress in prevention and treatment of caries and periodontal diseases has been translated to better oral health and improved tooth retention in the adult population. The ageing population and the increasing expectations of good oral health-related quality of life in older age pose formidable challenges to clinical care and healthcare systems. AIMS: The objective of this workshop was to critically review scientific evidence and develop specific recommendations to: (i) prevent tooth loss and retain oral function through prevention and treatment of caries and periodontal diseases later in life and (ii) increase awareness of the health benefits of oral health as an essential component of healthy ageing. METHODS: Discussions were initiated by three systematic reviews covering aspects of epidemiology of caries and periodontal diseases in elders, the impact of senescence on caries and periodontal diseases and the effectiveness of interventions. Recommendations were developed based on evidence from the systematic reviews and expert opinion. RESULTS: Key messages included: (i) the ageing population, trends in risk factors and improved tooth retention point towards an expected increase in the total burden of disease posed by caries and periodontal diseases in the older population; (ii) specific surveillance is required to monitor changes in oral health in the older population; (iii) senescence impacts oral health including periodontitis and possibly caries susceptibility; (iv) evidence indicates that caries and periodontal diseases can be prevented and treated also in older adults; (v) oral health and functional tooth retention later in life provides benefits both in terms of oral and general quality of life and in terms of preventing physical decline and dependency by fostering a healthy diet; (vi) oral healthcare professionals and individuals should not base decisions impacting tooth retention on chronological age but on level of dependency, life expectancy, frailty, comfort and quality of life; and (vii) health policy should remove barriers to oral health care for vulnerable elders. CONCLUSIONS: Consensus was reached on specific actionable priorities for public health officials, oral healthcare professionals, educators and workforce planners, caregivers and relatives as well as for the public and ageing patients. Some priorities have major implications for policymakers as health systems need to adapt to the challenge by systemwide changes to enable (promote) tooth retention later in life and management of deteriorating oral health in increasingly dependent elders.

Functional Isoeugenol-Modified Nanogel Coatings for the Design of Biointerfaces.

A new route for the synthesis of functional aqueous nanogels decorated with a controlled amount of surface-drafted isoeugenol molecules has been developed. Obtained nanogels exhibit two key functions: a) antibacterial activity against different oral pathogens and b) cell-adhesive and -growth-promoting properties. Functional nanogels can be potentially used as building blocks in the design of bioactive coatings on various implants preventing infections and accelerating tissue regeneration.

Analysis of Bacterial Activity in Sound and Cariogenic Biofilm: A Pilot in vivo Study.

Dental caries is a multifactorial disease with many associated microbial taxa, but only a few are notably contributing to acidogenicity. The ribosome number and the corresponding 16S ribosomal RNA (rRNA) concentration are considered a molecular indicator for general metabolic activity of bacteria, as they are elevated with increased anabolic and catabolic activities. We hypothesize that the activity of aciduric/acidogenic bacterial taxa, reflected by a rise in ribosomal counts, could resolve differences between plaque biofilm from sound surfaces and caries lesions. The included subjects were allocated to two groups: caries-free (CF) or caries-active (CA). CF subjects presented one donor site, namely one sound surface (CFS, n = 10), whereas CA subjects presented two donor sites: a cavitated lesion with an ICDAS score of 5-6 (CAC, n = 13), and a sound reference surface (CAS, n = 13). Four aciduric/acidogenic bacterial taxa (Streptococcus mutans, lactobacilli, Bifidobacterium dentium, and Scardovia wiggsiae) and one asaccharolytic taxon (fusobacteria) as a contrast were selected. 16S rRNA and 16S rRNA genes were quantified by quantitative PCR. Based on these parameters, bacterial and ribosomal counts, as well as relative activities were calculated as the quotient of relative ribosomal abundance and relative genome abundance. Caries-associated bacteria showed the highest relative activity in caries lesions (e.g. lactobacilli CAC: 177.5 ± 46.0%) and lower activities on sound surfaces (e.g. lactobacilli CAS: 96.3 ± 31.5%), whereas asaccharolytic fusobacteria were most active on sound surfaces and less active in caries lesions (CFS: 275.7 ± 171.1%; CAS: 205.8 ± 114.3%; CAC: 51.1 ± 19.0%). Thus, the present study suggests different activity patterns for biofilms from CF and CA individuals.

Trends in antibiotic use and microbial diagnostics in periodontal treatment: comparing surveys of German dentists in a ten-year period.

OBJECTIVES: The use of antibiotics and microbial tests in periodontal treatment among German dental practitioners was investigated in 2012-2013 and compared with 2002-2003 data. MATERIALS AND METHODS: One thousand four hundred representative German practitioners received a postal questionnaire requesting their prescribing habits concerning type, dose, frequency, and sequence of antibiotics adjunctive to mechanical debridement. Additionally, the use of local antimicrobials and microbial tests were recorded. RESULTS: The response rate was 29.1 % (407 reports). Drug combinations, especially amoxicillin plus metronidazole, were prescribed most frequently (32.8 %) with an increase of 7.4 % during the past decade, followed by clindamycin (29.3 %). Amoxicillin monotherapy was used unexpectedly frequently (17.0 %) and doxycycline (2.8 %) very infrequently. Then, 24.7 % prescribed antibiotics prior to mechanical therapy, while most dentists followed the recommended sequence. The use of local antimicrobials increased by 6.2 % and of microbial diagnostics by 20.8 %. CONCLUSIONS: Positive trends regarding position-paper-conform prescribing habits including the scheduling of systemic antibiotics and increasing use of local antimicrobials and microbial tests were observed. However, deficits and malpractice still exist in German practices. Unexpected is the widespread and increasing use of clindamycin. Continuing educational campaigns and strictly expressed real guidelines are needed. CLINICAL RELEVANCE: Indication and choice of antibiotic agents in causal periodontal therapy among German dentists have changed between 2003 and 2013 toward a more position-paper-based concept, but inappropriate prescriptions of second choice antibiotics still remain conspicuous.

Aciduric microbial taxa including Scardovia wiggsiae and Bifidobacterium spp. in caries and caries free subjects.

Actinobacteria came into focus of being potential caries-associated pathogens and could, together with the established Streptococcus mutans and lactobacilli thus function as caries indicator species. Here we analyzed the role and diagnostic predictive value of the acidogenic-aciduric species Scardovia wiggsiae and Bifidobacterium dentium together with S. mutans, lactobacilli and bifidobacteria in biofilm of non-cavitated (n = 20) and cavitated (n = 6) caries lesions versus controls (n = 30). For the genus Bifidobacterium and for B. dentium new sets of primers were designed. Based on real-time quantitative PCR and confirmed by DNA sequencing we found a higher prevalence (61.5%) of S. wiggsiae in caries lesions than in controls (40%). However, among the controls we found three individuals with both the highest absolute and relative S. wiggsiae numbers. Testing for S. mutans revealed the same prevalence as S. wiggsiae in caries lesions (61.5%) but in controls its prevalence was only 10%. B. dentium was never found in healthy plaque but in 30.8% of clinical cases, with the highest numbers in cavitated lesions. The Bifidobacterium-genus specific PCR had less discriminative power as more control samples were positive. We calculated the relative abundances and applied receiver operating characteristic analyses. The top results of specificity (93% and 87%) and sensitivity (100% and 88%) were found when the constraint set was "Lactobacillus relative abundance ≥0.02%" and "two aciduric species with a relative abundance of each ≥0.007%". Combinatory measurement of several aciduric taxa may be useful to reveal caries activity or even to predict caries progression.

Global analysis of saliva as a source of bacterial genes for insights into human population structure and migration studies.

BACKGROUND: The genetic diversity of the human microbiome holds great potential for shedding light on the history of our ancestors. Helicobacter pylori is the most prominent example as its analysis allowed a fine-scale resolution of past migration patterns including some that could not be distinguished using human genetic markers. However studies of H. pylori require stomach biopsies, which severely limits the number of samples that can be analysed. By focussing on the house-keeping gene gdh (coding for the glucose-6-phosphate dehydrogenase), on the virulence gene gtf (coding for the glucosyltransferase) of mitis-streptococci and on the 16S-23S rRNA internal transcribed spacer (ITS) region of the Fusobacterium nucleatum/periodonticum-group we here tested the hypothesis that bacterial genes from human saliva have the potential for distinguishing human populations. RESULTS: Analysis of 10 individuals from each of seven geographic regions, encompassing Africa, Asia and Europe, revealed that the genes gdh and ITS exhibited the highest number of polymorphic sites (59% and 79%, respectively) and most OTUs (defined at 99% identity) were unique to a given country. In contrast, the gene gtf had the lowest number of polymorphic sites (21%), and most OTUs were shared among countries. Most of the variation in the gdh and ITS genes was explained by the high clonal diversity within individuals (around 80%) followed by inter-individual variation of around 20%, leaving the geographic region as providing virtually no source of sequence variation. Conversely, for gtf the variation within individuals accounted for 32%, between individuals for 57% and among geographic regions for 11%. This geographic signature persisted upon extension of the analysis to four additional locations from the American continent. Pearson correlation analysis, pairwise Fst-cluster analysis as well as UniFrac analyses consistently supported a tree structure in which the European countries clustered tightly together and branched with American countries and South Africa, to the exclusion of Asian countries and the Congo. CONCLUSION: This study shows that saliva harbours protein-coding bacterial genes that are geographically structured, and which could potentially be used for addressing previously unresolved human migration events.

CLSM-Guided Imaging for Quantifying Endodontic Disinfection.

Elimination of microbes in the root canal system is crucial for achieving long-term success in endodontic treatment. Further efforts in study design and standardization are needed in order to improve the validity and comparability of in vitro results on endodontic disinfection procedures, in turn improving clinical outcomes. This study optimizes two models at all steps: tooth selection, pretreatment, inoculation method (by growth or centrifugation), and confocal laser scanning microscopy (CLSM)-guided imaging of LIVE/DEAD-stained specimens. Individual anatomical conditions lead to substantial differences in penetration depth. Sclerosis grading (SCG), a classification system introduced in this study, provides information about the sclerosis status of the dentine and is helpful for careful, specific, and comparable tooth selection in in vitro studies. Sonically activated EDTA for the pretreatment of roots, inoculation of Enterococcus faecalis in an overflow model, 3-4 weeks of incubation, as well as polishing of dentine slices before staining, led to advances in the visualization of bacterial penetration and irrigation depths. In contrast, NaOCl pretreatment negatively affected performance reproducibility and should be avoided in any pretreatment. Nonsclerotized teeth (SCG0) can be used for microbial semilunar-shaped inoculation by centrifugation as a "quick-and-dirty" model for initial orientation. In conclusion, CLSM-guided imaging for quantifying endodontic infection/disinfection is a very powerful method after the fine-tuning of materials and methods.

Genetic variability of mutans streptococci revealed by wide whole-genome sequencing.

BACKGROUND: Mutans streptococci are a group of bacteria significantly contributing to tooth decay. Their genetic variability is however still not well understood. RESULTS: Genomes of 6 clinical S. mutans isolates of different origins, one isolate of S. sobrinus (DSM 20742) and one isolate of S. ratti (DSM 20564) were sequenced and comparatively analyzed. Genome alignment revealed a mosaic-like structure of genome arrangement. Genes related to pathogenicity are found to have high variations among the strains, whereas genes for oxidative stress resistance are well conserved, indicating the importance of this trait in the dental biofilm community. Analysis of genome-scale metabolic networks revealed significant differences in 42 pathways. A striking dissimilarity is the unique presence of two lactate oxidases in S. sobrinus DSM 20742, probably indicating an unusual capability of this strain in producing H2O2 and expanding its ecological niche. In addition, lactate oxidases may form with other enzymes a novel energetic pathway in S. sobrinus DSM 20742 that can remedy its deficiency in citrate utilization pathway.Using 67 S. mutans genomes currently available including the strains sequenced in this study, we estimates the theoretical core genome size of S. mutans, and performed modeling of S. mutans pan-genome by applying different fitting models. An "open" pan-genome was inferred. CONCLUSIONS: The comparative genome analyses revealed diversities in the mutans streptococci group, especially with respect to the virulence related genes and metabolic pathways. The results are helpful for better understanding the evolution and adaptive mechanisms of these oral pathogen microorganisms and for combating them.

Prevalence and Phylogenetic Analysis of Lipoprotein-Gene ragB-1 of Porphyromonas gingivalis-A Pilot Study.

Porphyromonas gingivalis (P.g.) is a key pathogen involved in periodontal diseases. The aim of this study was to investigate the prevalence and phylogenetic origin of the lipoprotein-gene ragB in its most virulent variant, ragB-1 (co-transcribed with ragA-1 as locus rag-1), in different P.g. strains collected worldwide. A total of 138 P.g. strains were analyzed for the presence of ragB-1 by pooled analysis and subsequently individual PCRs. Sequencing a core fragment of ragB-1 of the individual strains made it possible to carry out a phylogenetic classification using sequence alignment. In total, 22 of the 138 P.g. strains tested positive for ragB-1, corresponding to a prevalence of 16%. The fragment investigated was highly conserved, with variations in the base sequence detected in only three strains (OMI 1072, OMI 1081, and OMI 1074). In two strains, namely OMI 1072 (original name: I-433) and OMI 1081 (original name: I-372), which originate from monkeys, two amino-acid alterations were apparent. Since ragB-1 has also been found in animal strains, it may be concluded that rag-1 was transferred from animals to humans and that this originally virulent variant was weakened by mutations over time so that new, less virulent, adapted commensal versions of rag (rag-2, -3, and -4), with P.g. as the host, evolved.