Annette Carola Anderson

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.

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.

Antimicrobial Effects of Dental Luting Glass Ionomer Cements on Streptococcus mutans

Objective. To reduce secondary caries, glass ionomer luting cements are often used for cementing of indirect restorations. This is because of their well-known antimicrobial potential through the release of fluoride ions. The aim of this in vitro study was to investigate the antimicrobial effect of five dental luting cements which were based on glass ionomer cement technology. Methods. Five different glass ionomer based luting cements were tested for their antimicrobial effects on Streptococcus mutans in two different experimental setups: (i) determination of colony-forming units (CFUs) in a plate-counting assay; (ii) live/dead staining (LDS) and fluorescence microscopy. All experiments were conducted with or without prior treatment of the materials using sterilized human saliva. Antimicrobial effects were evaluated for adherent and planktonic bacteria. Bovine enamel slabs (BES) were used as negative control. BES covered with 0.2% chlorhexidine (CHX) served as positive control. Results. Each of the tested materials significantly reduced the number of initially adhered CFUs; this reduction was even more pronounced after prior incubation in saliva. Antimicrobial effects on adherent bacteria were confirmed by live-dead staining. Conclusion. All five luting cements showed an antimicrobial potential which was increased by prior incubation with human saliva, suggesting an enhanced effect in vivo.

Antimicrobial Photoinactivation Using Visible Light Plus Water-Filtered Infrared-A (VIS + wIRA) Alters In Situ Oral Biofilms.

Recently, growing attention has been paid to antimicrobial photodynamic therapy (aPDT) in dentistry. Changing the microbial composition of initial and mature oral biofilm by aPDT using visible light plus water-filtered infrared-A wavelengths (VIS + wIRA) has not yet been investigated. Moreover, most aPDT studies have been conducted on planktonic bacterial cultures. Therefore, in the present clinical study we cultivated initial and mature oral biofilms in six healthy volunteers for 2 hours or 3 days, respectively. The biofilms were treated with aPDT using VIS+wIRA (200 mW cm-2), toluidine blue (TB) and chlorine e6 (Ce6) for 5 minutes. Chlorhexidine treated biofilm samples served as positive controls, while untreated biofilms served as negative controls. After aPDT treatment the colony forming units (CFU) of the biofilm samples were quantified, and the surviving bacteria were isolated in pure cultures and identified using MALDI-TOF, biochemical tests and 16S rDNA-sequencing. aPDT killed more than 99.9% of the initial viable bacterial count and 95% of the mature oral biofilm in situ, independent of the photosensitizer. The number of surviving bacterial species was highly reduced to 6 (TB) and 4 (Ce6) in the treated initial oral biofilm compared to the 20 different species of the untreated biofilm. The proportions of surviving bacterial species were also changed after TB- and Ce6-mediated aPDT of the mature oral biofilm, resulting in a shift in the microbial composition of the treated biofilm compared to that of the control biofilm. In conclusion, aPDT using VIS + wIRA showed a remarkable potential to eradicate both initial and mature oral biofilms, and also to markedly alter the remaining biofilm. This encourages the clinical use of aPDT with VIS + wIRA for the treatment of periimplantitis and periodontitis.

Shift of microbial composition of peri-implantitis-associated oral biofilm as revealed by 16S rRNA gene cloning

Purpose. Micro‐organisms are important triggers of peri‐implant inflammation and analysing their diversity is necessary for peri‐implantitis treatment. This study aimed to analyse and compare the microbiota associated with individuals with peri‐implantitis, as well as clinically healthy implant sites. Methodology. Subgingival biofilm samples were taken from 10 individuals with peri‐implantitis and from at least 1 clinically healthy implant. DNA was extracted and bacterial 16S rRNA genes were amplified using universal primers. After cloning the PCR‐products, amplified inserts of positive clones were digested using restriction endonucleases, and the chosen clones were sequenced. The 16S rDNA‐sequences were compared to those from the public sequence databases GenBank, EMBL and DDBJ to determine the corresponding taxa. Results. Differing distributions of taxa belonging to the phyla Firmicutes, Bacteroidetes, Fusobacteria, Actinobacteria, Proteobacteria, Synergistetes, Spirochaetae and TM 7 were detected in both the healthy implant (HI) and the peri‐implantitis (PI) groups. A significantly higher relative abundance of phylum Bacteroidetes, as well as of the species Fusobacterium nucleatum, were found in the PI group (P<0.05). The putative periodontal red complex (Porphyromonas gingivalis, Tannerella forsythia) was also detected at significantly higher levels in the PI group (P<0.05), whereas the yellow group, as well as the species Veillonella dispar, tended to be associated with the HI group. Conclusion. A shift in the healthy subgingival microbiota was shown in peri‐implantitis‐associated biofilm. Anaerobic Gram‐negative periopathogens, including P. gingivalis and T. forsythia, seem to play an important role in peri‐implantitis development and should be considered in treatment and prevention strategies.

In-vivo shift of the microbiota in oral biofilm in response to frequent sucrose consumption

Caries is associated with shifts of microbiota in dental biofilms and primarily driven by frequent sucrose consumption. Data on environmentally induced in vivo microbiota shifts are scarce therefore we investigated the influence of frequent sucrose consumption on the oral biofilm. Splint systems containing enamel slabs were worn for 3 × 7 days with 7-day intervals to obtain oral biofilm samples. After a three-month dietary change of sucking 10 g of sucrose per day in addition to the regular diet, biofilm was obtained again at the end of the second phase. The microbiota was analysed using Illumina MiSeq amplicon sequencing (v1-v2 region). In addition, roughness of the enamel surface was measured with laser scanning microscopy. The sucrose phase resulted in significant differences in beta-diversity and significantly decreased species richness. It was marked by a significant increase in abundance of streptococci, specifically Streptococcus gordonii, Streptococcus parasanguinis and Streptococcus sanguinis. Enamel surface roughness began to increase, reflecting initial impairment of dental enamel surface. The results showed that frequent sucrose consumption provoked compositional changes in the microbiota, leading to an increase of non-mutans streptococci, hence supporting the extended ecological plaque hypothesis and emphasizing the synergy of multiple bacterial species in the development of caries.