Disruption of Enterococcus Faecalis biofilms using individual and plasma polymer encapsulated D-amino acids.

OBJECTIVE: Our aim was to assess the anti-biofilm ability of previously unverified individual D-amino acids (DAAs), to produce plasma polymer encapsulated DAAs (PPEDAAs), to measure the shell thickness and subsequent release of DAAs, and to assess the effects of PPEDAAs on Enterococcus faecalis biofilms. MATERIALS AND METHODS: Microtitre tray assays were used to evaluate the effect of individual DAAs (D-leucine, D-methionine, D-tryptophan, and D-tyrosine) on E. faecalis biofilms of different maturity. A mixture and individual DAAs were encapsulated with a plasma polymer for 10, 20, 40, and 60 min. The shell thickness of PPEDAAs was analyzed by ultra-high-resolution scanning electron microscopy. The release of DAAs from the PPEDAAs encapsulated for 60 min was measured over 7 days using high-performance liquid chromatography. Static biofilms were used to assess the effect of PPEDAAs on E. faecalis biofilms. RESULTS: Individual DAAs reduced biofilm formation to various degrees, according to the DAA and the experimental times. The shell thicknesses of the PPEDAAs ranged between 31 and 76 nm and increased with encapsulation time. Diffusion of DAAs from the PPEDAAs occurred over 60 min for encapsulated D-leucine, D-methionine, and D-tyrosine and up to 7 days for D-tryptophan. PPEDAAs disrupted biofilms at every experimental time. CONCLUSIONS: PPEDAAs of various shell thickness can be produced with the proposed methodology, DAAs are subsequently released, and the anti-biofilm activity remains unaltered. CLINICAL RELEVANCE: Individual DAAs and PPEDAAs have anti-biofilm ability and can be considered as part of a biological strategy in endodontics.

Development of an in vitro biofilm model of the human supra-gingival microbiome for Oral microbiome transplantation.

The high prevalence of dental caries and periodontal disease place a significant burden on society, both socially and economically. Recent advances in genomic technologies have linked both diseases to shifts in the oral microbiota - a community of >700 bacterial species that live within the mouth. The development of oral microbiome transplantation draws on the success of fecal microbiome transplantation for the treatment of gut pathologies associated with disease. Many current in vitro oral biofilm models have been developed but do not fully capture the complexity of the oral microbiome which is required for successful OMT. To address this, we developed an in vitro biofilm system that maintained an oral microbiome with 252 species on average over 14 days. Six human plaque samples were grown in 3D printed flow cells on hydroxyapatite discs using artificial saliva medium (ASM). Biofilm composition and growth were monitored by high throughput sequencing and confocal microscopy/SEM, respectively. While a significant drop in bacterial diversity occurred, up to 291 species were maintained in some flow cells over 14 days with 70% viability grown with ASM. This novel in vitro biofilm model represents a marked improvement on existing oral biofilm systems and provides new opportunities to develop oral microbiome transplant therapies.

A proteomic investigation of Fusobacterium nucleatum alkaline-induced biofilms.

BACKGROUND: The Gram negative anaerobe Fusobacterium nucleatum has been implicated in the aetiology of periodontal diseases. Although frequently isolated from healthy dental plaque, its numbers and proportion increase in plaque associated with disease. One of the significant physico-chemical changes in the diseased gingival sulcus is increased environmental pH. When grown under controlled conditions in our laboratory, F. nucleatum subspecies polymorphum formed mono-culture biofilms when cultured at pH 8.2. Biofilm formation is a survival strategy for bacteria, often associated with altered physiology and increased virulence. A proteomic approach was used to understand the phenotypic changes in F. nucleatum cells associated with alkaline induced biofilms. The proteomic based identification of significantly altered proteins was verified where possible using additional methods including quantitative real-time PCR (qRT-PCR), enzyme assay, acidic end-product analysis, intracellular polyglucose assay and Western blotting. RESULTS: Of 421 proteins detected on two-dimensional electrophoresis gels, spot densities of 54 proteins varied significantly (p < 0.05) in F. nucleatum cultured at pH 8.2 compared to growth at pH 7.4. Proteins that were differentially produced in biofilm cells were associated with the functional classes; metabolic enzymes, transport, stress response and hypothetical proteins. Our results suggest that biofilm cells were more metabolically efficient than planktonic cells as changes to amino acid and glucose metabolism generated additional energy needed for survival in a sub-optimal environment. The intracellular concentration of stress response proteins including heat shock protein GroEL and recombinational protein RecA increased markedly in the alkaline environment. A significant finding was the increased abundance of an adhesin, Fusobacterial outer membrane protein A (FomA). This surface protein is known for its capacity to bind to a vast number of bacterial species and human epithelial cells and its increased abundance was associated with biofilm formation. CONCLUSION: This investigation identified a number of proteins that were significantly altered by F. nucleatum in response to alkaline conditions similar to those reported in diseased periodontal pockets. The results provide insight into the adaptive mechanisms used by F. nucleatum biofilms in response to pH increase in the host environment.

Polycationic Silver Nanoclusters Comprising Nanoreservoirs of Ag+ Ions with High Antimicrobial and Antibiofilm Activity.

Silver-based nano-antibiotics are rapidly developing as promising alternatives to conventional antibiotics. Ideally, to remain potent against a wide range of drug-resistant and anaerobic bacteria, silver-based nano-antibiotics should easily penetrate through the bacterial cell walls and actively release silver ions. In this study, highly monodispersed, ultrasmall (<3 nm), polycationic silver nanoclusters (pAgNCs) are designed and synthesized for the elimination of a range of common Gram-negative and Gram-positive pathogens and their corresponding established and matured biofilms, including those composed of multiple species. The pAgNCs also show greatly enhanced antibacterial efficacy against anaerobic bacteria such as Fusobacterium nucleatum and Streptococcus sanguinis. These results demonstrate that the cationic nature facilitates better penetration to the bacterial cell membrane while the presence of a high percentage (>50%) of silver ions (i.e., Ag+ nanoreservoirs) on the cluster surface maintains their efficiency in both aerobic and anaerobic conditions. Significantly, the pAgNCs showed a strong capacity to significantly delay the development of bacterial resistance when compared to similar-sized negatively charged silver nanoparticles or conventional antibiotics. This study demonstrates a novel design strategy that can lay the foundation for the development of future highly potent nano-antibiotics effective against a broad spectrum of pathogens and biofilms needed in many everyday life applications and industries.

Effect of alkaline growth pH on the expression of cell envelope proteins in Fusobacterium nucleatum.

Fusobacterium nucleatum is a Gram-negative anaerobic organism that plays a central role in the development of periodontal diseases. The progression of periodontitis is associated with a rise in pH of the gingival sulcus which promotes the growth and expression of virulence factors by periodontopathic bacteria. We have previously reported that the expression of specific cytoplasmic proteins is altered by a shift in growth pH. In the present study we have compared cell envelope protein expression of F. nucleatum during chemostat growth at pH 7.2 and 7.8. From a total of 176 proteins resolved from the cell envelope, 15 were found to have altered expression in response to an increase in growth pH and were identified by MS. Upregulated proteins included an outer membrane porin which has been identified as playing a role in virulence, a periplasmic chaperone which assists in the folding of outer membrane proteins, and a transporter thought to be involved with iron uptake. Proteins downregulated at pH 7.8 were consistent with our previous findings that the bacterium reduces its catabolism of energy-yielding substrates in favour of energy-storage pathways. Among the downregulated proteins, two transporters which are involved in the uptake of C4 dicarboxylates and phosphate were identified. A putative protease and an enzyme associated with the metabolism of glutamate were also identified. A high proportion of the cell envelope proteins suggested by these data to play a role in the organism's response to alkaline growth pH may have arisen by lateral gene transfer. This would support the hypothesis that genes that provide an ability to adapt to the changing conditions of the oral environment may be readily shared between oral bacteria.

Antimicrobial properties of calcium hydroxide dressing when used for long-term application: A systematic review.

This review aims to evaluate the antimicrobial efficacy of calcium hydroxide against endodontic pathogens when used for 7 days or longer. A systematic electronic literature search was performed in the PubMed, Embase and EBSCO Dentistry & Oral Sciences Source databases using appropriate key words to identify investigations written in the English language that examined the association between the contact time of intracanal calcium hydroxide dressing and its antimicrobial properties. There were no exclusions based on study design. The search yielded 6993 publications. After duplicate removal, 5913 publications were identified and 11 studies met the inclusion criteria. Results showed that the antimicrobial effect of calcium hydroxide for contact times ranging between seven and 45 days is comparable. Two studies demonstrated contradictory findings when exposure was extended to more than 45 days. Future studies are warranted to investigate and optimise calcium hydroxide application for longer periods and identify the potential benefits of its use in clinical settings.

D-amino acids reduce Enterococcus faecalis biofilms in vitro and in the presence of antimicrobials used for root canal treatment.

Enterococcus faecalis is the most frequent species present in post-treatment disease and plays a significant role in persistent periapical infections following root canal treatment. Its ability to persist in stressful environments is inter alia, due to its ability to form biofilms. The presence of certain D-amino acids (DAAs) has previously been shown to reduce formation of Bacillus subtilis biofilms. The aims of this investigation were to determine if DAAs disrupt biofilms in early and late growth stages for clinical E. faecalis strains and to test their efficacy in disrupting E. faecalis biofilms grown in sub-minimum inhibitory concentrations of commonly used endodontic biocides. From thirty-seven E. faecalis strains, the ten "best" biofilm producers were used to test the ability of a mixture containing D-leucine, D-methionine, D-tyrosine and D-tryptophan to reduce biofilm growth over a period of 24, 72 and 144 hours and when compared to their cognate L-Amino Acids (LAAs). We have previously shown that sub-MIC levels of tetracycline and sodium hypochlorite promotes biofilm growth in clinical strains of E. faecalis. DAAs were therefore tested for their effectiveness to reduce biofilm growth in the presence of sub-minimal concentrations of sodium hypochlorite (NaOCl-0.031%) and Odontocide™ (0.25% w/v), and in the presence of Odontopaste™ (0.25% w/v). DAAs significantly reduced biofilm formation for all strains tested in vitro, while DAAs significantly reduced biofilm formation compared to LAAs. The inhibitory effect of DAAs on biofilm formation was concentration dependent. DAAs were also shown to be effective in reducing E. faecalis biofilms in the presence of Odontopaste™ and sub-MIC levels of NaOCl and Odontocide™. The results suggest that the inclusion of DAAs into current endodontic procedures may reduce E. faecalis biofilms.

Investigation of the Cell Surface Proteome of Human Periodontal Ligament Stem Cells.

The present study examined the cell surface proteome of human periodontal ligament stem cells (PDLSC) compared to human fibroblasts. Cell surface proteins were prelabelled with CyDye before processing to extract the membrane lysates, which were separated using 2D electrophoresis. Selected differentially expressed protein "spots" were identified using Mass spectrometry. Four proteins were selected for validation: CD73, CD90, Annexin A2, and sphingosine kinase 1 previously associated with mesenchymal stem cells. Flow cytometric analysis found that CD73 and CD90 were highly expressed by human PDLSC and gingival fibroblasts but not by keratinocytes, indicating that these antigens could be used as potential markers for distinguishing between mesenchymal cells and epithelial cell populations. Annexin A2 was also found to be expressed at low copy number on the cell surface of human PDLSC and gingival fibroblasts, while human keratinocytes lacked any cell surface expression of Annexin A2. In contrast, sphingosine kinase 1 expression was detected in all the cell types examined using immunocytochemical analysis. These proteomic studies form the foundation to further define the cell surface protein expression profile of PDLSC in order to better characterise this cell population and help develop novel strategies for the purification of this stem cell population.

Abnormal pregnancy outcomes in mice using an induced periodontitis model and the haematogenous migration of Fusobacterium nucleatum sub-species to the murine placenta.

OBJECTIVES: To investigate if there is subspecies specific migration to the placenta by Fusobacterium nucleatum (Fn) and to determine whether experimentally induced periodontitis results in adverse pregnancy outcomes (APO) in mice. METHODS: Periodontitis was induced in pregnant mice using an inoculum of Fn and Porphyromonas gingivalis. In parallel, four sub-species of Fn were individually injected into the circulatory system. At day 18 of gestation, the placenta, liver, spleen and blood were harvested and litter size, number of viable fetuses and resorptions, maternal, fetal and placenta weights were recorded. For the direct inoculation group, some mice were allowed to deliver for assessment of length of gestation, litter size, maternal, placental and pup weight. The presence of Fn was assessed by PCR and inflammatory mediators were measured by ELISA or multiplex analysis. RESULTS: Mice with alveolar bone loss, a marker of periodontitis, demonstrated significantly higher fetal weights (p = 0.015) and fetal/placental weight ratios (p = 0.030). PCR analysis of maternal organs did not identify Fn in any extracted tissues. In mice that received direct injection of Fn subspecies, varying degrees of APO were observed including preterm birth, intrauterine growth restriction, and fetal loss. Haematogenous spread of only Fn subsp. nucleatum to the placenta was confirmed. Litter size was significantly smaller (p = 0.023) and the number of resorptions was higher in inoculated versus control groups. Mice injected with subsp. nucleatum had significantly increased circulating CRP levels (p = 0.020) compared to controls while the mice with induced periodontitis had increased levels of IL-6 (p = 0.047) and IL-8 (p = 0.105). CONCLUSIONS: Periodontitis in mice elevated fetal weight and the fetal weight/placental weight ratio. This study found that subsp. nucleatum migrated haematogenously to the placenta, leading to APO in mice. The study supports the potential role of Fn in the association between periodontitis and APO.

The effect of sodium hypochlorite on Enterococcus faecalis when grown on dentine as a single- and multi-species biofilm.

Enterococcus faecalis is often involved in the aetiology of apical periodontitis after endodontic treatment. This project aimed to establish, on dentine in vitro, a multi-species biofilm containing E. faecalis, and to determine if the organism had an increased resistance to sodium hypochlorite compared with an axenic biofilm. Biofilms were established on dentine discs in flow cells with either E. faecalis alone (axenic) or together with Fusobacterium nucleatum and Streptococcus sanguinis. Following treatment with either 0.9% sodium hypochlorite or saline, the viability of E. faecalis was determined by serial plating and qualitative analysis was performed by scanning electron microscopy and confocal laser scanning microscopy. Viable counts indicated that 0.9% NaOCl is highly effective against E. faecalis grown alone and as part of a multi-species biofilm (P = 0.0005 and P = 0.001, respectively). No significant difference in its survival in the two biofilm types was found (P = 0.8276).

Qualitative comparison of sonic or laser energisation of 4% sodium hypochlorite on an Enterococcus faecalis biofilm grown in vitro.

The effectiveness of sonic activation, laser activation and syringe irrigation of 4% sodium hypochlorite in removing an Enterococcus faecalis biofilm was compared. Biofilms were grown in extracted human single rooted teeth using a flow cell apparatus. After 4 weeks' growth, teeth were subjected to each treatment using 4% sodium hypochlorite and radicular dentinal surfaces of the root canals were analysed by scanning electron microscopy. Results showed that sonic activation and syringe irrigation with sodium hypochlorite showed reduced numbers of bacterial cells on the radicular dentine but were not effective in eliminating E. faecalis in the dentinal tubules. Laser activation of sodium hypochlorite resulted in clean dentine walls and undetectable levels of bacteria within dentinal tubules. Qualitatively, sonic or laser activation of 4% NaOCl resulted in greater bacterial reduction compared with syringe irrigation, with laser activation producing the greatest overall reduction.

Proteomic characterization of mesenchymal stem cell-like populations derived from ovine periodontal ligament, dental pulp, and bone marrow: analysis of differentially expressed proteins.

Postnatal mesenchymal stem/stromal-like cells (MSCs) including periodontal ligament stem cells (PDLSCs), dental pulp stem cells (DPSCs), and bone marrow stromal cells (BMSCs) are capable of self-renewal and differentiation into multiple mesenchymal cell lineages. Despite their similar expression of MSC-associated and osteoblastic markers, MSCs retain the capacity to generate structures resembling the microenvironments from which they are derived in vivo and represent a promising therapy for the regeneration of complex tissues in the clinical setting. With this in mind, systematic approaches are required to identify the differential protein expression patterns responsible for lineage commitment and mediating the formation of these complex structures. This is the first study to compare the differential proteomic expression profiles of ex vivo-expanded ovine PDLSCs, DPSCs, and BMSCs derived from an individual donor. The two-dimensional electrophoresis was performed and regulated proteins were identified by liquid chromatography--electrospray-ionization tandem mass spectrometry (MS and MS/MS), database searching, and de novo sequencing. In total, 58 proteins were differentially expressed between at least 2 MSC populations in both sheep, 12 of which were up-regulated in one MSC population relative to the other two. In addition, the regulation of selected proteins was also conserved between equivalent human MSC populations. We anticipate that differential protein expression profiling will provide a basis for elucidating the protein expression patterns and molecular cues that are crucial in specifying the characteristic growth and developmental capacity of dental and non-dental tissue-derived MSC populations. These expression patterns can serve as important tools for the regeneration of particular tissues in future stem cell-based tissue engineering studies using animal models.

Inability to form a biofilm of Streptococcus mutans on silver fluoride- and potassium iodide-treated demineralized dentin.

OBJECTIVE: The presence of a biofilm is necessary for both initiation and progression of dental caries. Silver-based preparations incorporated into, or applied onto, various materials designed for medical use have been shown to be effective in inhibiting biofilm formation. The purpose of this in vitro study was to measure whether a topical application of diamine silver fluoride (AgF) followed by potassium iodide (KI) on partially demineralized dentin affected the formation of a Streptococcus mutans biofilm. METHOD AND MATERIALS: Forty partially demineralized dentin disks were divided into 4 groups as follows: 10 disks as a control, 10 disks treated with AgF followed by KI, 10 disks treated with KI, and 10 disks treated with AgF. The outer surfaces of the disks were examined with a scanning electron microscope. Cross sections of the disks were subjected to electron probe microanalysis (EPMA) to determine the levels of calcium, phosphorous, silver, and fluoride in the dentin. RESULTS: An S mutans biofilm covered the entire exposed surfaces of all control and KI-treated disks. No discernible bacterial biofilm was detected on disks treated with AgF or AgF/KI. Detectable amounts of silver and fluoride were found up to 450 microm in the AgF and AgF/KI sections. CONCLUSIONS: Demineralized dentin disks treated with AgF and AgF/KI prevented the formation of an S mutans biofilm and were significantly more resistant to further demineralization than the control and KI-treated disks over the experimental period. The presence of silver and fluoride in the outer layers of the disks treated with AgF and AgF/KI was the likely cause of the prevention of biofilm formation. Additional studies are required before any clinical recommendations can be made.

Co-adhesion and biofilm formation by Fusobacterium nucleatum in response to growth pH.

Fusobacterium nucleatum is a Gram-negative anaerobic organism considered to play an important role in the progression of periodontal disease and is commonly found in clinical infections of other body sites. Apart from its metabolic versatility, its cell-surface properties enable it to attach to epithelial cells, collagen, gingival epithelial cells and other bacterial genera, but not with other Fusobacteria. The development of periodontitis is associated with a rise in pH in the gingival sulcus to around 8.5, and this is thought to occur by the catabolism of proteins supplied by gingival crevicular fluid. F. nucleatum is commonly isolated from diseased sites and has also been shown to survive in root canal systems at pH 9.0 after Ca(OH)(2) treatment. In order to survive hostile environmental conditions, such as nutrient deprivation and fluctuating temperature and pH, bacteria form biofilms, which are usually made up of multi-species co-aggregates. We have grown F. nucleatum in a chemostat at a growth rate consistent with that of oral bacteria in vivo and report that, at a growth pH of 8.2, F. nucleatum co-adheres and forms a homogeneous biofilm. Cell-surface hydrophobicity was determined in planktonic and co-adhering cells to characterise the interfacial interactions associated with the response to pH. Cell-surface hydrophobicity was found to increase at pH 8.2 and this was also associated with a decrease in the levels of intracellular polyglucose (IP) and an observed change in the bacterial cell morphology. To our knowledge, these results represent the first study in which F. nucleatum has been shown to co-adhere and form a biofilm, which may be important in the organism's persistence during the transition from health to disease in vivo.

The proteomic profile of Fusobacterium nucleatum is regulated by growth pH.

Fusobacterium nucleatum is a saccharolytic Gram-negative anaerobic organism believed to play an important role in the microbial succession associated with the development of periodontal disease. Its genome contains niche-specific genes shared with the other inhabitants of dental plaque, which may help to explain its ability to survive and grow in the changing environmental conditions experienced in the gingival sulcus during the transition from health to disease. The pH of the gingival sulcus increases during the development of periodontitis and this is thought to occur by the metabolism of nutrients supplied by gingival crevicular fluid. In comparison with other plaque inhabitants, F. nucleatum has the greatest ability to neutralize acidic environments. The differential expression of soluble cytoplasmic proteins induced by acidic (pH 6.4) or basic (pH 7.4 and 7.8) conditions, during long-term anaerobic growth in a chemostat, was identified by two-dimensional gel electrophoresis and image analysis software. Twenty-two proteins, found to have altered expression in response to external pH, were identified by tryptic digestion and mass spectrometry. Eight differentially expressed proteins associated with increased energy (ATP) production via the 2-oxoglutarate and Embden-Meyerhof pathways appeared to be directed towards either cellular biosynthesis or the maintenance of internal homeostasis. Overall, these results represent the first proteomic investigation of F. nucleatum and the identification of gene products which may be important in the organism's persistence during the transition from health to disease in vivo.