Lamprini Karygianni

New Bacterial Composition in Primary and Persistent/Secondary Endodontic Infections with Respect to Clinical and Radiographic Findings

INTRODUCTION The aim of the present study was to analyze the microbiota of primary and secondary/persistent endodontic infections of patients undergoing endodontic treatment with respect to clinical and radiographic findings. METHODS Samples from the root canals of 21 German patients were taken using 3 sequential sterile paper points. In the case of a root canal filling, gutta-percha was removed with sterile files, and samples were taken using sterile paper points. The samples were plated, and microorganisms were then isolated and identified morphologically by biochemical analysis and sequencing the 16S rRNA genes of isolated microorganisms. RESULTS In 12 of 21 root canals, 33 different species could be isolated. Six (50%) of the cases with isolated microorganisms were primary, and 6 (50%) cases were endodontic infections associated with root-filled teeth. Twelve of the isolated species were facultative anaerobic and 21 obligate anaerobic. Monomicrobial infections were found for Enterococcus faecalis and Actinomyces viscosus. E. faecalis was most frequently isolated in secondary endodontic infections (33%). Moraxella osloensis was isolated from a secondary endodontic infection that had an insufficient root canal filling accompanied by a mild sensation of pain. A new bacterial composition compromising Atopobium rimae, Anaerococcus prevotii, Pseudoramibacter alactolyticus, Dialister invisus, and Fusobacterium nucleatum was recovered from teeth with chronic apical abscesses. CONCLUSIONS New bacterial combinations were found and correlated to clinical and radiographic findings, particularly to chronic apical abscesses. M. osloensis was detected in root canals for the second time and only in German patients.

Comprehensive Analysis of Secondary Dental Root Canal Infections: A Combination of Culture and Culture-Independent Approaches Reveals New Insights

Persistence of microorganisms or reinfections are the main reasons for failure of root canal therapy. Very few studies to date have included culture-independent methods to assess the microbiota, including non-cultivable microorganisms. The aim of this study was to combine culture methods with culture-independent cloning methods to analyze the microbial flora of root-filled teeth with periradicular lesions. Twenty-one samples from previously root-filled teeth were collected from patients with periradicular lesions. Microorganisms were cultivated, isolated and biochemically identified. In addition, ribosomal DNA of bacteria, fungi and archaea derived from the same samples was amplified and the PCR products were used to construct clone libraries. DNA of selected clones was sequenced and microbial species were identified, comparing the sequences with public databases. Microorganisms were found in 12 samples with culture-dependent and -independent methods combined. The number of bacterial species ranged from 1 to 12 in one sample. The majority of the 26 taxa belonged to the phylum Firmicutes (14 taxa), followed by Actinobacteria, Proteobacteria and Bacteroidetes. One sample was positive for fungi, and archaea could not be detected. The results obtained with both methods differed. The cloning technique detected several as-yet-uncultivated taxa. Using a combination of both methods 13 taxa were detected that had not been found in root-filled teeth so far. Enterococcus faecalis was only detected in two samples using culture methods. Combining the culture-dependent and –independent approaches revealed new candidate endodontic pathogens and a high diversity of the microbial flora in root-filled teeth with periradicular lesions. Both methods yielded differing results, emphasizing the benefit of combined methods for the detection of the actual microbial diversity in apical periodontitis.

Comparison of the Bacterial Composition and Structure in Symptomatic and Asymptomatic Endodontic Infections Associated with Root-Filled Teeth Using Pyrosequencing

Residual microorganisms and/or re-infections are a major cause for root canal therapy failure. Understanding of the bacterial content could improve treatment protocols. Fifty samples from 25 symptomatic and 25 asymptomatic previously root-filled teeth were collected from Sudanese patients with periradicular lesions. Amplified 16S rRNA gene (V1-V2) variable regions were subjected to pyrosequencing (FLX 454) to determine the bacterial profile. Obtained quality-controlled sequences from forty samples were classified into 741 operational taxonomic units (OTUs) at 3% dissimilarity, 525 at 5% dissimilarity and 297 at 10% dissimilarity, approximately corresponding to species-, genus- and class levels. The most abundant phyla were: Firmicutes (29.9%), Proteobacteria (26.1%), Actinobacteria (22.72%), Bacteroidetes (13.31%) and Fusobacteria (4.55%). Symptomatic patients had more Firmicutes and Fusobacteria than asymptomatic patients, while asymptomatic patients showed more Proteobacteria and Actinobacteria. Interaction of disease status and age was observed by two-way ANOSIM. Canonical correspondence analysis for age, tooth restoration and disease status showed a correlation of disease status with the composition and prevalence of different members of the microbial community. The pyrosequencing analysis revealed a distinctly higher diversity of the microbiota compared to earlier reports. The comparison of symptomatic and asymptomatic patients showed a clear association of the composition of the bacterial community with the presence and absence of symptoms in conjunction with the patients’ age.

Enterococcus faecalis from Food, Clinical Specimens, and Oral Sites: Prevalence of Virulence Factors in Association with Biofilm Formation

Enterococci have gained significance as the cause of nosocomial infections; they occur as food contaminants and have also been linked to dental diseases. E. faecalis has a great potential to spread virulence as well as antibiotic resistance genes via horizontal gene transfer. The integration of food-borne enterococci into the oral biofilm in-vivo has been observed. Therefore, we investigated the virulence determinants and antibiotic resistance of 97 E. faecalis isolates from the oral cavity, food, and clinical specimens. In addition, phenotypic expression of gelatinase and cytolysin were tested, in-vitro biofilm formation was quantified and isolates were compared for strain relatedness via pulsed field gel electrophoresis (PFGE). Each isolate was found to possess two or more virulence genes, most frequently gelE, efaA, and asa1. Notably, plaque/saliva isolates possessed the highest abundance of virulence genes, the highest levels of phenotypic gelatinase and hemolysin activity and concurrently a high ability to form biofilm. The presence of asa1 was associated with biofilm formation. The biofilm formation capacity of clinical and plaque/saliva isolates was considerably higher than that of food isolates and they also showed similar antibiotic resistance patterns. These results indicate that the oral cavity can constitute a reservoir for virulent E. faecalis strains possessing antibiotic resistance traits and at the same time distinct biofilm formation capabilities facilitating exchange of genetic material.

Rapid species-level identification of vaginal and oral lactobacilli using MALDI-TOF MS analysis and 16S rDNA sequencing

BackgroundLactobacillus represents a large genus with different implications for the human host. Specific lactobacilli are considered to maintain vaginal health and to protect from urogenital infection. The presence of Lactobacillus species in carious lesions on the other hand is associated with progressive caries. Despite their clinical significance, species-level identification of lactobacilli still poses difficulties and mostly involves a combination of different phenotypic and genotypic methods. This study evaluated rapid MALDI-TOF MS analysis of vaginal and oral Lactobacillus isolates in comparison to 16S rDNA analysis.ResultsBoth methods were used to analyze 77 vaginal and 21 oral Lactobacillus isolates. The concordance of both methods was at 96% with five samples discordantly identified. Fifteen different Lactobacillus species were found in the vaginal samples, primarily L. iners, L. crispatus, L. jensenii and L. gasseri. In the oral samples 11 different species were identified, mostly L. salivarius, L. gasseri, L. rhamnosus and L. paracasei. Overall, the species found belonged to six different phylogenetic groups. For several samples, MALDI-TOF MS analysis only yielded scores indicating genus-level identification. However, in most cases the species found agreed with the 16S rDNA analysis result.ConclusionMALDI-TOF MS analysis proved to be a reliable and fast tool to identify lactobacilli to the species level. Even though some results were ambiguous while 16S rDNA sequencing yielded confident species identification, accuracy can be improved by extending reference databases. Thus, mass spectra analysis provides a suitable method to facilitate monitoring clinically relevant Lactobacillus species.

Ultrasonic activation and chemical modification of photosensitizers enhances the effects of photodynamic therapy against Enterococcus faecalis root-canal isolates

BACKGROUND The aim of this study was to evaluate the effect of photodynamic therapy (PDT) on Enterococcus faecalis biofilms in artificially infected root canals using modified photosensitizers and passive ultrasonic activation. METHODS Two hundred and seventy extracted human teeth with one root canal were instrumented utilizing ProTaper files, autoclaved, infected with E. faecalis T9 for 72 h and divided into different groups: irrigation with 3% sodium hypochlorite (NaOCl), 20% ethylenediaminetetraacetic acid (EDTA), or 20% citric acid, PDT without irrigation, PDT accompanied by irrigation with NaOCl, EDTA, or citric acid, PDT using an EDTA-based photosensitizer or a citric-acid-based photosensitizer and PDT with ultrasonic activation of the photosensitizer. A 15 mg/ml toluidine blue served as the photosensitizer, activated by a 100 mW LED light source. Sterile paper points were used for sampling the root canals and dentin chips were collected to assess the remaining contamination after treatment. Samples were cultured on blood agar plates and colony forming units were quantified. RESULTS PDT alone achieved a reduction in E. faecalis counts by 92.7%, NaOCl irrigation alone and combined with PDT by 99.9%. The antibacterial effects increased by the combination of irrigation using EDTA or citric acid and PDT compared to irrigation alone. More than 99% of E. faecalis were killed using PDT with the modified photosensitizers and ultrasonic activation. CONCLUSIONS NaOCl based disinfection achieved the highest antimicrobial effect. Using PDT with an EDTA-based or citric-acid-based phozosensitizer or activating the photosensitizer with ultrasound resulted in a significantly higher reduction in E. faecalis counts compared to conventional PDT.

Antimicrobial photodynamic therapy using visible light plus water-filtered infrared-A (wIRA).

The aim of this study was to investigate the effectiveness of antimicrobial photodynamic therapy (APDT) using visible light together with water-filtered infrared-A (VIS+wIRA) to eradicate single species of planktonic bacteria and micro-organisms during initial oral bacterial colonization in situ. A broadband VIS+wIRA radiator with a water-filtered spectrum in the range 580-1400 nm was used for irradiation. Toluidine blue (TB) was utilized as a photosensitizer at concentrations of 5, 10, 25 and 50 µg ml(-1). The unweighted (absolute) irradiance was 200 mW cm(-2) and it was applied for 1 min. Planktonic cultures of Streptococcus mutans and Enterococcus faecalis were treated with APDT. Salivary bacteria harvested by centrifugation of native human saliva were also tested. In addition, initial bacterial colonization of bovine enamel slabs carried in the mouths of six healthy volunteers was treated in the same way. Up to 2 log(10) of S. mutans and E. faecalis were killed by APDT. Salivary bacteria were eliminated to a higher extent of 3.7-5 log(10). All TB concentrations tested proved to be highly effective. The killing rate of bacteria in the initial oral bacterial colonization was significant (P=0.004) at all tested TB concentrations, despite the interindividual variations found among study participants. This study has shown that APDT in combination with TB and VIS+wIRA is a promising method for killing bacteria during initial oral colonization. Taking the healing effects of wIRA on human tissue into consideration, this technique could be helpful in the treatment of peri-implantitis and periodontitis.

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

ABSTRACT 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.

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

ABSTRACT 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.

Initial Bacterial Adhesion on Different Yttria-Stabilized Tetragonal Zirconia Implant Surfaces in Vitro

Bacterial adhesion to implant biomaterials constitutes a virulence factor leading to biofilm formation, infection and treatment failure. The aim of this study was to examine the initial bacterial adhesion on different implant materials in vitro. Four implant biomaterials were incubated with Enterococcus faecalis, Staphylococcus aureus and Candida albicans for 2 h: 3 mol % yttria-stabilized tetragonal zirconia polycrystal surface (B1a), B1a with zirconium oxide (ZrO2) coating (B2a), B1a with zirconia-based composite coating (B1b) and B1a with zirconia-based composite and ZrO2 coatings (B2b). Bovine enamel slabs (BES) served as control. The adherent microorganisms were quantified and visualized using scanning electron microscopy (SEM); DAPI and live/dead staining. The lowest bacterial count of E. faecalis was detected on BES and the highest on B1a. The fewest vital C. albicans strains (42.22%) were detected on B2a surfaces, while most E. faecalis and S. aureus strains (approximately 80%) were vital overall. Compared to BES; coated and uncoated zirconia substrata exhibited no anti-adhesive properties. Further improvement of the material surface characteristics is essential.