Effect of thymoquinone on Fusobacterium nucleatum­associated biofilm and inflammation.

Periodontitis affects oral tissues and induces systemic inflammation, which increases the risk of cardiovascular disease and metabolic syndrome. Subgingival plaque accumulation is a trigger of periodontitis. Fusobacterium nucleatum (FN) contributes to subgingival biofilm complexity by intercalating with early and late bacterial colonizers on tooth surfaces. In addition, inflammatory responses to FN are associated with the progression of periodontitis. Nigella sativa Lin. seed, which is known as black cumin (BC), has been used as a herbal medicine to treat ailments such as asthma and infectious diseases. The current study examined the inhibitory effect of BC oil and its active constituents, thymol (TM) and thymoquinone (TQ), on FN­associated biofilm and inflammation. FN­containing biofilms were prepared by co­cultivation with an early dental colonizer, Actinomyces naeslundii (AN). The stability and biomass of FN/AN dual species biofilms were significantly higher compared with FN alone. This effect was retained even with prefixed cells, indicating that FN/AN co­aggregation is mediated by physicochemical interactions with cell surface molecules. FN/AN biofilm formation was significantly inhibited by 0.1% TM or TQ. Confocal laser scanning microscopy indicated that treatment of preformed FN/AN biofilm with 0.01% of BC, TM or TQ significantly reduced biofilm thickness, and TQ demonstrated a cleansing effect equivalent to that of isopropyl methylphenol. TQ dose­dependently suppressed TNF­α production from a human monocytic cell line, THP­1 exposed to FN, yet showed no toxicity to THP­1 cells. These results indicated that oral hygiene care using TQ could reduce FN­associated biofilm and inflammation in gingival tissue.

Forward Genetic Dissection of Biofilm Development by Fusobacterium nucleatum: Novel Functions of Cell Division Proteins FtsX and EnvC.

Fusobacterium nucleatum is a key member of the human oral biofilm. It is also implicated in preterm birth and colorectal cancer. To facilitate basic studies of fusobacterial virulence, we describe here a versatile transposon mutagenesis procedure and a pilot screen for mutants defective in biofilm formation. Out of 10 independent biofilm-defective mutants isolated, the affected genes included the homologs of the Escherichia coli cell division proteins FtsX and EnvC, the electron transport protein RnfA, and four proteins with unknown functions. Next, a facile new gene deletion method demonstrated that nonpolar, in-frame deletion of ftsX or envC produces viable bacteria that are highly filamentous due to defective cell division. Transmission electron and cryo-electron microscopy revealed that the ΔftsX and ΔenvC mutant cells remain joined with apparent constriction, and scanning electron microscopy (EM) uncovered a smooth cell surface without the microfolds present in wild-type cells. FtsX and EnvC proteins interact with each other as well as a common set of interacting partners, many with unknown function. Last, biofilm development is altered when cell division is blocked by MinC overproduction; however, unlike the phenotypes of ΔftsX and ΔenvC mutants, a weakly adherent biofilm is formed, and the wild-type rugged cell surface is maintained. Therefore, FtsX and EnvC may perform novel functions in Fusobacterium cell biology. This is the first report of an unbiased approach to uncover genetic determinants of fusobacterial biofilm development. It points to an intriguing link among cytokinesis, cell surface dynamics, and biofilm formation, whose molecular underpinnings remain to be elucidated.IMPORTANCE Little is known about the virulence mechanisms and associated factors in F. nucleatum, due mainly to the lack of convenient genetic tools for this organism. We employed two efficient genetic strategies to identify F. nucleatum biofilm-defective mutants, revealing FtsX and EnvC among seven biofilm-associated factors. Electron microscopy established cell division defects of the ΔftsX and ΔenvC mutants, accompanied with a smooth cell surface, unlike the microfold, rugged appearance of wild-type bacteria. Proteomic studies demonstrated that FtsX and EnvC interact with each other as well as a set of common and unique interacting proteins, many with unknown functions. Importantly, blocking cell division by MinC overproduction led to formation of a weakly adherent biofilm, without alteration of the wild-type cell surface. Thus, this work links cell division and surface dynamics to biofilm development and lays a foundation for future genetic and biochemical investigations of basic cellular processes in this clinically significant pathogen.

Antibacterial and antibiofilm activities of docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) against periodontopathic bacteria.

Docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) are two major omega-3 polyunsaturated fatty acids (n-3 PUFAs) with antimicrobial properties. In this study, we evaluated the potential antibacterial and antibiofilm activities of DHA and EPA against two periodontal pathogens, Porphyromonas gingivalis (P. gingivalis) and Fusobacterium nucleatum (F. nucleatum). MTT assay showed that DHA and EPA still exhibited no cytotoxicity to human oral tissue cells when the concentration came to 100 µM and 200 µM, respectively. Against P. gingivalis, DHA and EPA showed the same minimum inhibitory concentration (MIC) of 12.5 µM, and a respective minimum bactericidal concentration (MBC) of 12.5 µM and 25 µM. However, the MIC and MBC values of DHA or EPA against F. nucleatum were both greater than 100 µM. For early-stage bacteria, DHA or EPA displayed complete inhibition on the planktonic growth and biofilm formation of P. gingivalis from the lowest concentration of 12.5 µM. And the planktonic growth of F. nucleatum was slightly but not completely inhibited by DHA or EPA even at the concentration of 100 µM, however, the biofilm formation of F. nucleatum at 24 h was significantly restrained by 100 µM EPA. For exponential-phase bacteria, 100 µM DHA or EPA completely killed P. gingivalis and significantly decreased the viable counts of F. nucleatum. Meanwhile, the morphology of P. gingivalis was apparently damaged, and the virulence factor gene expression of P. gingivalis and F. nucleatum was strongly downregulated. Besides, the viability and the thickness of mature P. gingivalis biofilm, together with the viability of mature F. nucleatum biofilm were both significantly decreased in the presence of 100 µM DHA or EPA. In conclusion, DHA and EPA possessed antibacterial activities against planktonic and biofilm forms of periodontal pathogens, which suggested that DHA and EPA might be potentially supplementary therapeutic agents for prevention and treatment of periodontal diseases.

Rose bengal uptake by E. faecalis and F. nucleatum and light-mediated antibacterial activity measured by flow cytometry.

Antibacterial photodynamic therapy (aPDT) using rose bengal (RB) and blue-light kills bacteria through the production of reactive oxygen derivates. However, the interaction mechanism of RB with bacterial cells remains unclear. This study investigated the uptake efficiency and the antibacterial activity of blue light-activated RB against Enterococcus faecalis and Fusobacterium nucleatum. Spectrophotometry and epifluorescence microscopy were used to evaluate binding of RB to bacteria. The antibacterial activity of RB after various irradiation times was assessed by flow cytometry in combination with cell sorting. Uptake of RB increased in a concentration dependent manner in both strains although E. faecalis displayed higher uptake values. RB appeared to bind specific sites located at the cellular poles of E. faecalis and at regular intervals along F. nucleatum. Blue-light irradiation of samples incubated with RB significantly reduced bacterial viability. After incubation with 10µM RB and 240s irradiation, only 0.01% (±0.01%) of E. faecalis cells and 0.03% (±0.03%) of F. nucleatum survived after treatment. This study indicated that RB can bind to E. faecalis and F. nucleatum in a sufficient amount to elicit effective aPDT. Epifluorescence microscopy showed a yet-unreported property of RB binding to bacterial membranes. Flow cytometry allowed the detection of bacteria with damaged membranes that were unable to form colonies on agars after cell sorting.

A transfer efficiency model for ultrasound mediated drug/gene transferring into cells.

Ultrasound is a very promising technology to mediated drug/gene transferring into cells. However the relations between cell experimental conditions and results have been still unknown. It seriously impeded the development of the technology. In the article, a transfer efficiency model for ultrasound mediated drug/gene transferring into cells in stable cavitation was constructed. To testify the model, the numerical results were compared with the cell experimental data from six literatures in the entirely different experimental conditions. The numerical results fit the cell experimental data well. Despite simplifications and limitations, the model for the first time established the relationship between the cell experimental results about transfer efficiency and the conditions including ultrasound, microbubble and cells in stable cavitation.

Morphological, biochemical, physiological and molecular aspects of the response of Fusobacterium nucleatum exposed to subinhibitory concentrations of antimicrobials.

Subinhibitory concentrations (SICs) of antimicrobials may result in alterations in bacterial biology with implications for its potential aggression. This has considerable importance for the resident microbiota. Our aim was to analyze the effects of SICs of antimicrobials on the morphological, biochemical, physiological and molecular characteristics of the resident anaerobic Fusobacterium nucleatum. Fourteen strains were obtained from F. nucleatum ATCC 25586, selected by culturing on SICs of ampicillin, ampicillin/sulbactam, clindamycin, chloramphenicol, levofloxacin, metronidazole and piperacillin/tazobactam and subsequent culturing in the absence of drugs. Antimicrobial susceptibility, bacterial morphology, biochemical profiles and biofilm formation were evaluated. Genotyping and analysis of protein profiles were also performed. The antimicrobial susceptibility patterns showed that most of the derived strains were less sensitive to the antimicrobials, even after culturing them without drugs. Morphological and cell complexity alterations were observed, mainly in strains grown in SICs of ß-lactam; these strains also expressed a reduced ability for biofilm formation. The other strains showed an increase in biofilm formation but no apparent morphological changes. Alterations were observed in the carbohydrate metabolism patterns and in the activity of microbial enzymes. Several proteins were positively or negatively regulated and there was polymorphism in the DNA from all derived strains. Therefore, SICs of antimicrobials induce alterations in F. nucleatum, which directly impact its biology. These results emphasize the risk of inadequate antibioticotherapy, which may have serious implications for clinical microbiology and infectious diseases and also may interfere with the host-bacteria relationship.

TLR2 sensing of F. nucleatum and S. sanguinis distinctly triggered gingival innate response.

Gingival tissue faces constant exposure to micro-organisms. It functions as part of the host response, an anti-microbial barrier that recognizes and discriminates between commensal and pathogenic bacteria. This study aimed to evaluate and compare the effects of cell wall extracts from different periodontal bacteria, commensals Streptococcus sanguinis and Fusobacterium nucleatum and the pathogen Porphyromonas gingivalis, on the innate immune response of gingival keratinocytes and the role of TLR2 in regulating this. We assayed mRNA levels to determine the expression of human beta-defensins (hbetaD2, hbetaD3), interleukin-1alpha, -1beta, 6 and 8 and matrix metalloproteinase-9. F. nucleatum extracts induced beta-defensin and inflammatory marker mRNA expression at higher levels than P. gingivalis. Extracts from the Gram-positive commensal S. sanguinis did not upregulate the host response. TLR2 extinction inhibited the upregulation of beta-defensin and cytokine transcripts by F. nucleatum extracts but, in contrast, led to a weak induction of hbetaD3 after challenge with S. sanguinis extracts. Although F. nucleatum strongly induces innate immune and inflammatory mediators, S. sanguinis limits their expression through TLR2. Together, our data demonstrate that gingival keratinocytes recognize and discriminate between Gram-positive and Gram-negative commensal extracts, in part through TLR2, to activate different signaling pathways of the innate immune host response.

Fiber optic fluorescence microprobe for endodontic diagnosis.

Successful endodontic therapy requires total debridement as well as complete obturation of the root canal to the cemento-dentinal junction. The goal of this study was to investigate the feasibility of using quantitative fluorescence spectroscopy for the detection and localization of pathological dentin, pulpal remnants, and microorganisms within the root canal. Specific aims were to identify: 1) characteristic excitation/emission spectra for healthy dentin, decayed dentin, enamel, and pulp; 2) the potential of specific spectral data for differentiating between these tissues; and 3) the potential of spectral data for detecting the presence and identifying four common endodontic pathogens. Fluorescence spectra were determined in the tissues of permanent human teeth, extirpated healthy and necrotic pulps, and four endodontic pathogens. Excitation/emission spectra were collected at 366 nm, 405 nm, and 440 nm excitation. Marked differences in spectral signatures between the different tissues under investigation were observed. We postulate that the differences in fluorescence spectra of decayed vs. healthy dentin are due to the loss of mineralized tissue components and increased organic presence and water in these tissues. Pulpal tissue showed distinctly different fluorescence spectra from healthy and decayed dentin, providing a basis for differentiating between tissue categories. Each bacterial species demonstrated distinct spectral emission patterns.

Phototoxic effect of visible light on Porphyromonas gingivalis and Fusobacterium nucleatum: an in vitro study.

The antibacterial effect of visible light irradiation combined with photosensitizers has been reported. The objective of this was to test the effect of visible light irradiation without photosensitizers on the viability of oral microorganisms. Strains of Porphyromonas gingivalis, Fusobacterium nucleatum, Streptococcus mutans and Streptococcus faecalis in suspension or grown on agar were exposed to visible light at wavelengths of 400-500 nm. These wavelengths are used to photopolymerize composite resins widely used for dental restoration. Three photocuring light sources, quartz-tungsten-halogen lamp, light-emitting diode and plasma-arc, at power densities between 260 and 1300 mW/cm2 were used for up to 3 min. Bacterial samples were also exposed to a near-infrared diode laser (wavelength, 830 nm), using identical irradiation parameters for comparison. The results show that blue light sources exert a phototoxic effect on P. gingivalis and F. nucleatum. The minimal inhibitory dose for P. gingivalis and F. nucleatum was 16-62 J/cm2, a value significantly lower than that for S. mutans and S. faecalis (159-212 J/cm2). Near-infrared diode laser irradiation did not affect any of the bacteria tested. Our results suggest that visible light sources without exogenous photosensitizers have a phototoxic effect mainly on Gram-negative periodontal pathogens.

Clonal persistence of oral Fusobacterium nucleatum in infancy.

Once established, early-colonizing bacterial species tend to persist in the mouth. To obtain detailed information on the population dynamics of early-colonizing oral anaerobes, we examined the clonal diversity and persistence of clones among oral Fusobacterium nucleatum populations during the first 2 yrs of life. Consecutive salivary samples from 12 infants, collected at 2, 6, 12, 18, and 24 mos of age, yielded a total of 546 F. nucleatum isolates for clonal typing with arbitrarily primed PCR (AP-PCR). Up to 7 AP-PCR types were simultaneously detected in each sample. In 11 out of the 12 infants examined, AP-PCR types persisted for up to 1 yr. Strain turnover rate was high during the first year of life, but then the occurrence of persistent clones increased. This study indicates a wide genetic diversity within the species and provides evidence for the increasing persistence of F. nucleatum clones in the oral cavity with age.

Evaluation of co-aggregation among Streptococcus mitis, Fusobacterium nucleatum and Porphyromonas gingivalis.

AIMS: To develop a semi-quantitative method for evaluating co-aggregation reactions among three bacterial species, and to examine the influence of Fusobacterium nucleatum on the adherence of Porphyromonas gingivalis. METHODS AND RESULTS: The method involves coating hydroxyapatite (HAP) discs with streptococcal cells and treatment with radio-labelled bacterial cell suspensions. The sensitivity of the method was estimated by comparison with a turbidometric co-aggregation assay. Results from the two methods were in close agreement. Streptococcus mitis-coated HAP discs were immersed in a 3H-labelled Fus. nucleatum cell suspension and then a 14C-labelled P. gingivalis cell suspension. The discs were then pyrolysed to recover and quantify the released 3H and 14C radioactivity. The number of Fus. nucleatum cells on the discs increased with immersion time and this, in turn, resulted in elevated adherence of P. gingivalis. CONCLUSION: The data indicate that the method closely reflects co-aggregation characters, and that Fus. nucleatum has a positive effect on the adherence of P. gingivalis. SIGNIFICANCE AND IMPACT OF THE STUDY: The present method, which is designed to mimic the oral environment, should prove useful in the semi-quantitative evaluation of co-aggregation reactions.

Kinetics of coaggregation of Porphyromonas gingivalis with Fusobacterium nucleatum using an automated microtiter plate assay.

Coaggregation between Porphyromonas gingivalis and Fusobacterium nucleatum strains was previously studied using either a semi-quantitative macroscopic assay or radioactive tracer assays. A new automated microtiter plate assay is introduced, in which the plate reader (Vmax) was adapted to allow quantitative evaluation of the kinetics of coaggregation. F nucleatum PK 1594 coaggregated with P. gingivalis HG 405 with a maximal coaggregation rate of 1.05 mOD/min, which occurred at a P. gingivalis to F. nucleatum cell ratio of 1 to 2. F. nucleatum PK 1594 failed to do so with P. gingivalis strains A 7436 or ATCC 33277. Galactose inhibition of this coaggregation could be quantitatively measured over a wide range of concentrations to demonstrate its dose-dependent manner. P. gingivalis HG 405 failed to coaggregate with F. nucleatum strains ATCC 25586 and ATCC 49256. The assay used in the present study is a sensitive and efficient quantitative automated tool to study coaggregation and may replace tedious radioactive tracer assays.