Isolation, characterization, and fibroblast uptake of bacterial extracellular vesicles from Porphyromonas gingivalis strains.

Periodontitis is an inflammatory condition caused by bacteria and represents a serious health problem worldwide as the inflammation damages the supporting tissues of the teeth and may predispose to systemic diseases. Porphyromonas gingivalis is considered a keystone periodontal pathogen that releases bacterial extracellular vesicles (bEVs) containing virulence factors, such as gingipains, that may contribute to the pathogenesis of periodontitis. This study aimed to isolate and characterize bEVs from three strains of P. gingivalis, investigate putative bEV uptake into human oral fibroblasts, and determine the gingipain activity of the bEVs. bEVs from three bacterial strains, ATCC 33277, A7A1-28, and W83, were isolated through ultrafiltration and size-exclusion chromatography. Vesicle size distribution was measured by nano-tracking analysis (NTA). Transmission electron microscopy was used for bEV visualization. Flow cytometry was used to detect bEVs and gingipain activity was measured with an enzyme assay using a substrate specific for arg-gingipain. The uptake of bEVs into oral fibroblasts was visualized using confocal microscopy. NTA showed bEV concentrations from 108 to 1011 particles/mL and bEV diameters from 42 to 356 nm. TEM pictures demonstrated vesicle-like structures. bEV-gingipains were detected both by flow cytometry and enzyme assay. Fibroblasts incubated with bEVs labeled with fluorescent dye displayed intracellular localization consistent with bEV internalization. In conclusion, bEVs from P. gingivalis were successfully isolated and characterized, and their uptake into human oral fibroblasts was documented. The bEVs displayed active gingipains demonstrating their origin from P. gingivalis and the potential role of bEVs in periodontitis.

Outcome of Endodontic Retreatment Using 2 Root Canal Irrigants and Influence of Infection on Healing as Determined by a Molecular Method: A Randomized Clinical Trial.

INTRODUCTION: This study compared the clinical and radiographic outcome of endodontic retreatment of teeth with apical periodontitis using either 1% sodium hypochlorite (NaOCl) or 2% chlorhexidine digluconate (CHX) as the irrigant. The influence of residual infection detected by a molecular method on the outcome was also examined. METHODS: Fifty-two root-filled teeth with apical periodontitis were randomly assigned into 2 groups according to the irrigant used during retreatment. Root canal microbiological samples taken before (S1) and after (S2) preparation using either NaOCl or CHX irrigation and after calcium hydroxide medication (S3) were subjected to 16S ribosomal RNA gene-based real-time quantitative polymerase chain reaction (qPCR) to quantify total bacteria. The periapical status was scored using the periapical index and dichotomized as healed (<3) or not healed (≥3) at the 1- and 4-year follow-up. RESULTS: Forty-five (NaOCl, n = 20; CHX, n = 25) and 33 teeth (NaOCl, n = 16; CHX, n = 17) were available at the 1- and 4-year follow-up, respectively. After 1 year, 65% in the NaOCl group and 64% in the CHX group healed, with no differences between them (P > .05). At the later follow-up, the corresponding figures were 81% and 82%, respectively (P > .05). Canals that yielded qPCR-negative results in S3 had a higher healing rate (79%) than qPCR-positive canals (45%, P < .05). The mean bacterial load increased from S2 to S3 in half of the unhealed cases (P < .05). All S3-positive canals containing <3.12 × 103 bacterial cell counts healed. Increasing the apical level of the root canal filling influenced the outcome (P < .05). CONCLUSIONS: No significant differences in the clinical outcome between 1% NaOCl and 2% CHX were found. Bacterial persistence at the time of filling as detected by qPCR significantly affected the outcome.

Structural and functional analyses of Rubisco from arctic diatom species reveal unusual posttranslational modifications.

The catalytic performance of the major CO2-assimilating enzyme, ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), restricts photosynthetic productivity. Natural diversity in the catalytic properties of Rubisco indicates possibilities for improvement. Oceanic phytoplankton contain some of the most efficient Rubisco enzymes, and diatoms in particular are responsible for a significant proportion of total marine primary production as well as being a major source of CO2 sequestration in polar cold waters. Until now, the biochemical properties and three-dimensional structures of Rubisco from diatoms were unknown. Here, diatoms from arctic waters were collected, cultivated, and analyzed for their CO2-fixing capability. We characterized the kinetic properties of five and determined the crystal structures of four Rubiscos selected for their high CO2-fixing efficiency. The DNA sequences of the rbcL and rbcS genes of the selected diatoms were similar, reflecting their close phylogenetic relationship. The Vmax and Km for the oxygenase and carboxylase activities at 25 °C and the specificity factors (Sc/o) at 15, 25, and 35 °C were determined. The Sc/o values were high, approaching those of mono- and dicot plants, thus exhibiting good selectivity for CO2 relative to O2 Structurally, diatom Rubiscos belong to form I C/D, containing small subunits characterized by a short ßA-ßB loop and a C-terminal extension that forms a ß-hairpin structure (ßE-ßF loop). Of note, the diatom Rubiscos featured a number of posttranslational modifications of the large subunit, including 4-hydroxyproline, ß-hydroxyleucine, hydroxylated and nitrosylated cysteine, mono- and dihydroxylated lysine, and trimethylated lysine. Our studies suggest adaptation toward achieving efficient CO2 fixation in arctic diatom Rubiscos.

Microbial Analysis of Endodontic Infections in Root-filled Teeth with Apical Periodontitis before and after Irrigation Using Pyrosequencing.

INTRODUCTION: The root canal microbiota in root-filled teeth with post-treatment apical periodontitis before and after chemomechanical instrumentation and irrigation with either 1% sodium hypochlorite (NaOCl) or 2% chlorhexidine digluconate were analyzed by using the pyrosequencing method. METHODS: Samples from 10 root-filled teeth with apical periodontitis undergoing retreatment were taken before (S1) and after (S2) preparation using irrigation with either NaOCl (n = 5) or 2% chlorhexidine digluconate (n = 5). DNA was extracted, and the 16S rRNA gene (V3-V5) variable regions were amplified and subjected to pyrosequencing (GS junior 454) to determine the bacterial composition. RESULTS: Pyrosequencing yielded 43,797 sequence reads in S1 and 9196 in S2 samples. Overall, 125 bacterial species belonging to 68 genera (S1, 59; S2, 38) and 9 phyla were found. The most abundant and prevalent phyla in S1 and S2 samples were Firmicutes, Fusobacteria, Bacteroidetes, and Actinobacteria. The most represented, abundant, and prevalent genera in S1 and S2 samples were Streptococcus and Fusobacterium. The most prevalent species in S1 and S2 samples were Fusobacterium nucleatum ss. vincentii, Streptococcus oralis/mitis, Streptococcus intermedius, and Streptococcus gordonii. The mean number of species per root canal was 20 (range, 4-37) in S1 and 9 (range, 4-15) in S2, respectively. CONCLUSIONS: A high interindividual diversity was observed in both S1 and S2 samples, with no difference between the two irrigation groups. F. nucleatum ss. vincentii and some Streptococcus species were the most prevalent species in pre-preparation and post-preparation samples during retreatment of root-filled teeth with infection.

Bacterial diversity in medication-related osteonecrosis of the jaw.

OBJECTIVE: The aim was to study the association between microflora and medication-related osteonecrosis of the jaw (MRONJ) by using culture-independent molecular techniques to detect bacteria in necrotic bone lesions. STUDY DESIGN: Included were 18 consecutive patients with MRONJ, 10 with osteoporosis and 8 cancer patients. Bone biopsies were retrieved from the center of the necrotic bone and from visually healthy bone, and 16 S rRNA gene fragments from bacterial DNA were amplified with polymerase chain reaction. RESULTS: The study revealed a diversity of bacteria represented by 16 S rRNA sequences in all the necrotic bone samples and in 60% of the visually healthy bone. Eight dominating taxa groups were identified at the genus level: Porphyromonas, Lactobacillus, Tannerella, Prevotella, Actinomyces, Treponema, Streptococcus, and Fusobacterium. CONCLUSIONS: The necrotic bone lesions contained mainly anaerobic bacteria, representative of periodontal microflora, suggesting that a periodontal infection in combination with antiresorptive treatment could initiate osteonecrosis.

Antibacterial Effectiveness of 2 Root Canal Irrigants in Root-filled Teeth with Infection: A Randomized Clinical Trial.

INTRODUCTION: This study compared the antibacterial effects of 1% sodium hypochlorite (NaOCl) and 2% chlorhexidine digluconate (CHX) during retreatment of teeth with apical periodontitis. METHODS: Root canal-treated teeth with apical periodontitis were randomly distributed into 2 groups. Bacteriological samples were taken from the canals before (S1) and after (S2) preparation using either NaOCl or CHX irrigation and after calcium hydroxide medication (S3); 16S ribosomal RNA gene-based real-time quantitative polymerase chain reaction was performed to quantify total bacteria, streptococci, and Enterococcus faecalis. RESULTS: Forty-nine teeth were available for analysis (NaOCl, n = 20; CHX, n = 29). Bacterial DNA occurred in all S1 samples, streptococci in 57% and E. faecalis in 6%. The total bacterial counts decreased from S1 to S2 in both groups (P < .01) but were higher in S3 than S2 (P < .01). Thirty-five percent of the teeth in the NaOCl group were positive in S2, decreasing to 20% in S3. In the CHX group, 41% were positive in S2, decreasing to 31% in S3. The bacterial load in S1 influenced the incidence of bacteria in S2 (P < .01). Streptococci were significantly reduced in both groups, and E. faecalis was found in only 1 S2 sample and not in S3. No significant difference between NaOCl and CHX was found. CONCLUSIONS: NaOCl and CHX both reduced bacterial counts and the number of infected canals. Intracanal medication with calcium hydroxide reduced the number of canals with persistent infection but resulted in overall larger bacterial counts in the cases positive for bacteria. The effectiveness of antimicrobial treatment can be influenced by the initial bacterial load.

Polymorphisms in the interleukin-10 gene and chronic periodontitis in patients with atherosclerotic and aortic aneurysmal vascular diseases.

BACKGROUND: Chronic periodontitis (CP), atherosclerotic and aortic aneurysmal vascular diseases (VD) are chronic inflammatory conditions with multifactorial etiologies, including involvement of predisposing genetic factors. In a previous study, polymorphisms in the gene for the anti-inflammatory interleukin-1 receptor antagonist were associated with CP in patients with VD. OBJECTIVE: This study investigates whether polymorphisms in the gene for the anti-inflammatory interleukin-10 (IL10) could be related to CP in the same manner. METHODS: Seventy-two patients with VD of whom 35 had CP were genotyped for single nucleotide polymorphisms (SNPs) in the IL10 -592 (rs1800872), -819 (rs1800871), and -1,082 (rs1800896) gene by Taqman rtPCR method and by DNA sequencing. RESULTS: The C alleles and C/C genotypes of IL10 -592 and IL10 -819 frequencies were significantly higher, while the frequencies of the IL10 -592 (C/A) and IL10 -819 (C/T) heterozygote genotypes were significantly lower in the VD group with CP compared to those without CP. The IL10 haplotype ATA frequency (-1,082, -819, -592) showed a trend to a significant difference between the two groups indicating protection against CP. CONCLUSIONS: Taken together, our findings suggest an independent association of genetic polymorphisms in the IL-10 gene locus with CP in patients with VD. Development of CP and the implications on vascular disease emphasize the importance of early detection and adequate treatment of periodontitis among these patients.

Diversity of Veillonella spp. from subgingival plaque by polyphasic approach.

In a biofilm such as the subgingival microflora, strain-specific properties or factors induced by the host may impart a survival advantage to some bacterial strains. Periodontal disease has been associated with chronic obstructive pulmonary disease (COPD) and we previously found high amounts of Veillonella in the subgingival microflora of COPD subjects. Differentiation of Veillonella is difficult. The aims of this study were to identify subgingival Veillonella isolates by phenotypic, genetic typing and molecular genetic methods, and further, to assess if Veillonella strain properties or identity correlated with periodontal disease or COPD. From 22 subjects, 26 subgingival Veillonella isolates and one pulmonary isolate were analysed. The majority of the subgingival Veillonella isolates were identified as Veillonella parvula. Genotyping showed heterogeneity within strains of the same species. A subgingival and pulmonary isolate in one COPD subject was found to be genetically identical strains of V. parvula. Scanning electron microscopy of the lung biopsy confirmed single small cocci adhering or coaggregating with larger cocci on the airway epithelium. Apart from a variation in cellular fatty acid composition of six subgingival isolates from periodontitis subjects, no correlation between the subgingival Veillonella strains or genotypes and the presence of either periodontitis or COPD was found. In conclusion, V. parvula was the predominant subgingival Veillonella species with high genetic variability within strains of the same species. Subgingival V. parvula can translocate to the lungs; however, Veillonella identity or genotype did not correlate with periodontal disease or COPD.