Vidar Bakken

Taxonomy, biology, and periodontal aspects of Fusobacterium nucleatum.

The pathogenic potential of Fusobacterium nucleatum and its significance in the development of periodontal diseases, as well as in infections in other organs, have gained new interest for several reasons. First, this bacterium has the potential to be pathogenic because of its number and frequency in periodontal lesions, its production of tissue irritants, its synergism with other bacteria in mixed infections, and its ability to form aggregates with other suspected pathogens in periodontal disease and thus act as a bridge between early and late colonizers on the tooth surface. Second, of the microbial species that are statistically associated with periodontal disease, F. nucleatum is the most common in clinical infections of other body sites. Third, during the past few years, new techniques have made it possible to obtain more information about F. nucleatum on the genetic level, thereby also gaining better knowledge of the structure and functions of the outer membrane proteins (OMPs). OMPs are of great interest with respect to coaggregation, cell nutrition, and antibiotic susceptibility. This review covers what is known to date about F. nucleatum in general, such as taxonomy and biology, with special emphasis on its pathogenic potential. Its possible relationship to other periodontal bacteria in the development of periodontal diseases and the possible roles played by OMPs are considered.

Caries experience and quantification of Streptococcus mutans and Streptococcus sobrinus in saliva of Sudanese schoolchildren.

Streptococcus mutans and Streptococcus sobrinus are among the most commonly isolated bacterial species implicated as etiological agents of dental caries. Details of the composition of the oral microflora related to dental caries should aid in assessing the prevalence and risk of disease at an individual level. The aim of the present study was to determine the presence and relative amounts of S. mutans and S. sobrinus in the saliva samples obtained from schoolchildren in Khartoum State, the Sudan, and to study the association of the amounts of S. mutans and S. sobrinus with caries experience, socioeconomic status and sugar-sweetened snacks in this population. 140 samples, 30 of which were from individuals with caries experience, were examined using quantitative real-time polymerase chain reaction (qRT-PCR) with specific oligonucleotide primers. The mean ratio of fold differences of S. mutans to S. sobrinus was 0.77 (SD 5.4) and 2.29 (SD 6.0) for samples obtained from caries-free and caries-active individuals, respectively. This suggested that the proportion of S. sobrinus was higher than S.mutans in the caries-active group when compared to the caries-free group. An association was found between children with caries-active lesions and the frequent consumption of sticky desserts and higher socioeconomic status. S. sobrinus seems to be associated with caries experience in the studied population. A proposal of caries screening programs designed to test for S. sobrinus in this population may be developed.

Ethnic Disparities in the Prevalence of Periodontitis Among High School Students in Sudan

BACKGROUND There are limited data on the epidemiology and risk factors of periodontitis in young populations in developing nations. This study assesses the prevalence of periodontal attachment loss and aggressive periodontitis and the association with ethnicity among high school students in Sudan. METHODS The study sample consisted of 1,200 students, 13 to 19 years old, selected from 38 public and private high schools using a multistage, stratified sampling design. The subjects were interviewed and examined clinically. Periodontal parameters were assessed at six sites per tooth. Subjects with aggressive periodontitis were identified. RESULTS A total of 3.4% of the subjects had aggressive periodontitis, and 16.3% and 8.2% of the subjects had at least one tooth with > or = 4 and > or = 5 mm attachment loss, respectively. A significantly higher percentage of subjects of African tribal ethnicity had attachment loss > or = 4 and > or = 5 mm compared to Afro-Arab tribes (19.8% versus 14.7%, P = 0.02; and 12% versus 6.4%, P = 0.004, respectively), and had a higher prevalence of aggressive periodontitis (6% versus 2.3%; P = 0.01) and higher risk of being diagnosed with this disease (odds ratio = 2.7; P <0.0001). African ethnicity was also associated with a significantly higher number of teeth with attachment loss than in Afro-Arabs (P <0.01). Comparison by gender showed a significantly higher percentage of males with aggressive periodontitis (4.9% versus 2%; P <0.01) and a higher risk for this disease (odds ratio = 2.5; P = 0.01) than in females. However, the prevalence of subjects with attachment loss > or = 4 and > or = 5 mm was comparable in the two gender groups. CONCLUSIONS Our results show that aggressive periodontitis is highly prevalent in this population. African ethnicity (versus Afro-Arab) and male gender were risk factors for aggressive periodontitis.

Purification and partial characterization of a major outer-membrane protein of Fusobacterium nucleatum

The major outer-membrane proteins of 40-41 kDa were identified by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) in Fusobacterium nucleatum strains ATCC 10953, ATCC 25586, F3, F6 and Fev1. The proteins were purified by preparative gel electrophoresis. Their behaviour in gel filtration and gel electrophoresis, their sensitivity to proteolytic enzymes, and their amino acid composition were investigated. The purified proteins were partly sequenced from the N-terminal end. A 36.5 kDa portion was protected against extrinsic proteolytic (trypsin, chymotrypsin or pronase) digestion of whole cells. This polypeptide was isolated and partially sequenced from the N-terminal end. From these data and data from extrinsic iodination it was concluded that the N-terminal end of the protein is probably exposed on the surface of the cell. A database search revealed amino acid sequence similarity in an Ala-Pro-rich region of outer-membrane protein A (OmpA) in other Gram-negative bacteria.

Fusobacterium nucleatum Enters Normal Human Oral Fibroblasts In Vitro

BACKGROUND Fusobacterium nucleatum, a commensal opportunistic oral bacterium, is capable of invading gingival epithelial cells, but the entrance into human primary oral fibroblast cells has not been documented. This study evaluated the ability of three strains of F. nucleatum (F. nucleatum ssp. nucleatum, F. nucleatum ssp. polymorphum, and F. nucleatum ssp. vincentii) to enter gingival fibroblasts (GFs) and periodontal ligament fibroblasts (PLFs). METHODS GFs and PLFs were cocultured for various periods of time with different strains of F. nucleatum. Scanning and transmission electron microscopy, together with confocal laser scanning microscopy, were used to visualize the entrance and presence of bacteria in host cells. Flow cytometry was performed to compare the load of internalized bacteria in GFs and PLFs exposed for 3 and 5 hours to live F. nucleatum labeled with fluorescein isothiocyanate. RESULTS All three strains of F. nucleatum were found entering and located in the cytoplasm of GFs and PLFs after 1 hour of exposure. Flow cytometry tests revealed a significant increase in the fluorescent signal, compared to baseline, derived from bacteria internalized in fibroblasts exposed for 3 hours (P <0.001); a further increase was found at 5 hours. The greatest bacterial mass in exposed fibroblasts of both types was of F. nucleatum ssp. polymorphum; the smallest was of F. nucleatum ssp. vincentii. Although not statistically significant, PLFs had a higher bacterial load than corresponding GFs. CONCLUSION F. nucleatum was capable of entering GFs and PLFs in a manner that is dependent on the cell type and the bacterial strain.

Outer membrane proteins of Fusobacterium nucleatum Fev1.

Outer membrane enriched material from six strains of Fusobacterium nucleatum was analysed by SDS-PAGE. The protein profiles of all the strains were dominated by proteins with molecular masses of about 40 kDa, and a very high degree of homology in relation to apparent molecular masses was observed. In all strains except Fev1, one of the most dominant proteins exhibited heat modifiable properties, having an apparent molecular mass of about 38 kDa and 42 kDa when heated in SDS at 50 and 100 degrees C, respectively. None of the proteins of the outer membrane of F. nucleatum Fev1 demonstrated such heat modifiable properties. The 40 kDa protein, and several other proteins, appear to be both exposed on the cell surface and peptidoglycan associated.

Prevalence of Aggregatibacter actinomycetemcomitans in Sudanese patients with aggressive periodontitis: a case–control study

BACKGROUND AND OBJECTIVE Aggregatibacter actinomycetemcomitans is considered a possible etiological agent for aggressive periodontitis. The aim of this study was to determine the prevalence of the JP2 clone and non-JP2 genotypes of A. actinomycetemcomitans in the subgingival plaque of patients with aggressive periodontitis and controls among Sudanese high-school students. MATERIAL AND METHODS In a previous study we examined a large representative sample of students attending high schools in Khartoum, Sudan. In this population, 17 patients with aggressive periodontitis and 17 controls (14-19 years of age) consented to participate in the present study. The subjects underwent a clinical periodontal examination, and subgingival dental plaque samples were collected using paper points. The presence of the A. actinomycetemcomitans JP2 clone and non-JP2 genotypes were assessed using loop-mediated isothermal amplification (LAMP) and the PCR. RESULTS The JP2 clone of A. actinomycetemcomitans was not detected in the subgingival plaque of either the cases or the controls. Non-JP2 types of A. actinomycetemcomitans were detected in the subgingival plaque of 12 (70.6%) patients with aggressive periodontitis and from only one (5.9%) control subject, showing a significantly higher frequency of detection in cases than in controls (p = 0.0001). The odds ratio for the detection of A. actinomycetemcomitans in the subgingival plaque of the patients with aggressive periodontitis was 38.4 (95% confidence interval: 4.0-373.0; p = 0.002). The PCR and LAMP methods showed identical results pertaining to the identification of non-JP2 types of A. actinomycetemcomitans. CONCLUSIONS The JP2 clone of A. actinomycetemcomitans was not detected in the subgingival plaque of high school subjects in Sudan. The detection of non-JP2 types of A. actinomycetemcomitans may be a useful marker of increased risk for development of aggressive periodontitis in young subjects.

Proteomic analysis of ampicillin-resistant oral Fusobacterium nucleatum.

INTRODUCTION Fusobacterium nucleatum represents one of the predominant anaerobic species in the oral microbiota. Penicillin-resistant F. nucleatum have been isolated from intra- and extraoral infections. This study aimed to assess ampicillin resistance in F. nucleatum by investigating the synthesis of resistance-associated proteins. METHODS Ampicillin-resistant and ampicillin-susceptible F. nucleatum isolates were obtained from 22 dental plaque samples. Two-dimensional gel electrophoresis and mass spectrometry were used to investigate bacterial protein synthesis. Proteins exhibiting statistically significant quantitative changes between sensitive and resistant isolates were identified using peptide mass mapping and matrix-assisted laser desorption/ionization - time of flight/time of flight (MALDI-TOF/TOF) mass spectrometry. RESULTS Twenty-three F. nucleatum isolates were recovered from plaque samples and their ampicillin minimum inhibitory concentrations ranged between 0.125 microg/ml and 256 microg/ml. Analysis of the bacterial cellular proteins by two-dimensional gel electrophoresis resolved 154-246 distinct protein spots (mean 212, n = 9). Between 32% and 83% of the protein spots were common for the F. nucleatum isolates. Comparisons of the protein profiles of sensitive and resistant isolates revealed the presence of a 29 kDa protein and significant increases in the synthesis of two proteins at 37 and 46 kDa in the ampicillin-resistant F. nucleatum isolates. These proteins were identified as a class D beta-lactamase, ATP-binding cassette (ABC) transporter ATP-binding protein and enolase, respectively. CONCLUSION Synthesis of a class D beta-lactamase by ampicillin-resistant F. nucleatum isolates could complicate antimicrobial treatment because these enzymes might confer resistance to many classes of beta-lactam antibiotics. The differences observed in protein synthesis between ampicillin-resistant and ampicillin-susceptible F. nucleatum may contribute to the antibiotic resistance and virulence of these bacteria.

Characterization of extracellular polymeric matrix, and treatment of Fusobacterium nucleatum and Porphyromonas gingivalis biofilms with DNase I and proteinase K.

Background : Biofilms are organized communities of microorganisms embedded in a self-produced extracellular polymeric matrix (EPM), often with great phylogenetic variety. Bacteria in the subgingival biofilm are key factors that cause periodontal diseases; among these are the Gram-negative bacteria Fusobacterium nucleatum and Porphyromonas gingivalis. The objectives of this study were to characterize the major components of the EPM and to test the effect of deoxyribonuclease I (DNase I) and proteinase K. Methods : F. nucleatum and P. gingivalis bacterial cells were grown in dynamic and static biofilm models. The effects of DNase I and proteinase K enzymes on the major components of the EPM were tested during biofilm formation and on mature biofilm. Confocal laser scanning microscopy was used in observing biofilm structure. Results : Proteins and carbohydrates were the major components of the biofilm matrix, and extracellular DNA (eDNA) was also present. DNase I and proteinase K enzymes had little effect on biofilms in the conditions used. In the flow cell, F. nucleatum was able to grow in partially oxygenated conditions while P. gingivalis failed to form biofilm alone in similar conditions. F. nucleatum supported the growth of P. gingivalis when they were grown together as dual species biofilm. Conclusion : DNase I and proteinase K had little effect on the biofilm matrix in the conditions used. F. nucleatum formed biofilm easily and supported the growth of P. gingivalis, which preferred anaerobic conditions.

Lymphangiogenesis Is Induced during Development of Periodontal Disease

Lymphangiogenesis, the formation of new lymphatics, is associated with chronic inflammation and tissue injury, and its role is to enhance lymphatic flow, immune cell transport, and antigen clearance. It is unknown if lymphangiogenesis takes place during periodontal disease development, and we hypothesized that growth of lymphatic vessels occurs in gingiva during development of periodontitis in mice. Inflammation was induced in gingiva with Porphyromonas gingivalis gavage, and bone resorption was verified after 42 days. Growth of lymphatic and blood vessels was measured after immunofluorescent staining with LYVE-1 and CD31. Expression of vascular endothelial growth factors and 2 inflammatory cytokines was investigated 10 days post-infection. Gingival lymphangiogenesis was found 10 days and 42 days post-infection, but proliferation of vessels was observed only in the shortest observation period. Epithelial expression of vascular growth factors (VEGF) A, C, and D was observed in gingiva, and increased numbers of immune cells expressing VEGF-C were found after infection, along with up-regulation of IL-1β and TNF-α at protein levels. We conclude that lymphangiogenesis takes place in gingiva during periodontal disease development, and that up-regulation of vascular growth factor C in recruited immune cells is likely important for the growth of lymphatic vessels.