Highlights • EGCG eradicated E. faecalis growth and biofilm. • EGCG suppressed the expression of key virulence genes of E. faecalis. • Antimicrobial action of EGCG occurred through the generation of ...hydroxyl radical.
In periodontal health, oral streptococci constitute up to 80% of the plaque biofilm. Yet, destructive inflammatory events of the periodontium are rare. This observation suggests that oral ...streptococci may possess mechanisms to co-exist with the host. However, the mechanisms employed by oral streptococci to modulate the innate immune response have not been well studied. One of the key virulence factors produced by oral streptococci is hydrogen peroxide (H
O
). In mammalian cells, H
O
triggers the activation of nuclear factor erythroid 2-related factor 2 (Nrf2), a key pathway mediating antioxidant defence. This study aimed to determine (1) if H
O
producing oral streptococci activated the Nrf2 pathway in macrophages, and (2) if the activation of Nrf2 influenced the innate immune response. We found that oral streptococci downregulated the innate immune response in a H
O
dependent manner through the activation of the Nrf2. The activation of the Nrf2 signalling pathway led to the inhibition of nuclear factor kappa-light-chain-enhancer of activated B cells (NFĸB), the key transcription factor regulating pro-inflammatory response. This study showed for the first time that oral streptococci are unlikely passive bystanders but could play an active role in the maintenance of periodontal health by preventing overt inflammation.
Plaque biofilm is the primary etiological agent of periodontal disease. Biofilm formation progresses through multiple developmental stages beginning with bacterial attachment to a surface, followed ...by development of microcolonies and finally detachment and dispersal from a mature biofilm as free planktonic bacteria. Tissue damage arising from inflammatory response to biofilm is one of the hallmark features of periodontal disease. A consequence of tissue damage is the release of ATP from within the cell into the extracellular space. Extracellular ATP (eATP) is an example of a danger associated molecular pattern (DAMP) employed by mammalian cells to elicit inflammatory and damage healing responses. Although, the roles of eATP as a signaling molecule in multi-cellular organisms have been relatively well studied, exogenous ATP also influences bacteria biofilm formation. Since plaque biofilms are continuously exposed to various stresses including exposure to the host damage factors such as eATP, we hypothesized that eATP, in addition to eliciting inflammation could potentially influence the biofilm lifecycle of periodontal associated bacteria. We found that eATP rather than nutritional factors or oxidative stress induced dispersal of
, an organism associated with periodontal disease. eATP induced biofilm dispersal through chelating metal ions present in biofilm. Dispersed
biofilm, regardless of natural or induced dispersal by exogenous ATP, were more adhesive and invasive compared to planktonic or biofilm counterparts, and correspondingly activated significantly more pro-inflammatory cytokine production in infected periodontal fibroblasts. Dispersed
also showed higher expression of
, a virulence factor implicated in adhesion and invasion, compared to planktonic or biofilm bacteria. This study revealed for the first time that periodontal bacterium is capable of co-opting eATP, a host danger signaling molecule to detach from biofilms. Our results further showed that dispersed
possessed distinct virulence characteristics compared to their biofilm and planktonic counterparts.
is the primary etiological agent of aggressive periodontal disease. Identification of novel virulence factors at the genome-wide level is hindered by lack of efficient genetic tools to perform ...mutagenesis in this organism. The
mariner transposon is known to yield a random distribution of insertions in an organism's genome with requirement for only a TA dinucleotide target and is independent of host-specific factors. However, the utility of this system in
is unknown. In this study, we found that
transposon mutagenesis occurs at a high frequency (×10
), and can be universally applied to wild-type
strains of serotypes a, b, and c. The
transposon inserts were stably inherited in
transconjugants in the absence of antibiotics. A library of 16,000 mutant colonies of
was screened for reduced biofilm formation. Mutants with transposon inserts in genes encoding pilus, putative ion transporters, multidrug resistant proteins, transcription regulators and enzymes involved in the synthesis of extracellular polymeric substance, bacterial metabolism and stress response were discovered in this screen. Our results demonstrated the utility of the
mutagenesis system as a novel genetic tool for functional genomic analysis in
.
Catechol-O-methyltransferase (COMT), an enzyme that metabolizes catecholamines, has recently been implicated in the modulation of pain. Our group demonstrated that human genetic variants of COMT are ...predictive for the development of Temporomandibular Joint Disorder (TMJD) and are associated with heightened experimental pain sensitivity (
Diatchenko et al. 2005
). Variants associated with heightened pain sensitivity produce lower COMT activity. Here we report the mechanisms underlying COMT-dependent pain sensitivity. To characterize the means whereby elevated catecholamine levels, resulting from reduced COMT activity, modulate heightened pain sensitivity, we administered a COMT inhibitor to rats and measured behavioral responsiveness to mechanical and thermal stimuli. We show that depressed COMT activity results in enhanced mechanical and thermal pain sensitivity. This phenomenon is completely blocked by the nonselective β-adrenergic antagonist propranolol or by the combined administration of selective β
2
- and β
3
-adrenergic antagonists, while administration of β
1
-adrenergic, α-adrenergic, or dopaminergic receptor antagonists fail to alter COMT-dependent pain sensitivity. These data provide the first direct evidence that low COMT activity leads to increased pain sensitivity via a β
2/3
-adrenergic mechanism. These findings are of considerable clinical importance, suggesting that pain conditions resulting from low COMT activity and/or elevated catecholamine levels can be treated with pharmacological agents that block both β
2
- and β
3
-adrenergic receptors.
Nanomaterials (NMs) such as titanium dioxide (nano-TiO2) and hydroxyapatite (nano-HA) are widely used in food, personal care, and many household products. Due to their extensive usage, the risk of ...human exposure is increased and may trigger NMs specific biological outcomes as the NMs interface with the cells. However, the interaction of nano-TiO2 and nano-HA with cells, their uptake and subcellular distribution, and the cytotoxic effects are poorly understood. Herein, we characterized and examined the cellular internalization, inflammatory response and cytotoxic effects of nano-TiO2 and nano-HA using TR146 human oral buccal epithelial cells as an in vitro model. We showed both types of NMs were able to bind to the cellular membrane and passage into the cells in a dose dependent manner. Strikingly, both types of NMs exhibited distinct subcellular distribution profile with nano-HA displaying a higher preference to accumulate near the cell membrane compared to nano-TiO2. Exposure to both types of NMs caused an elevated reactive oxygen species (ROS) level and expression of inflammatory transcripts with increasing NMs concentration. Although cells treated with nano-HA induces minimal apoptosis, nano-TiO2 treated samples displayed approximately 28% early apoptosis after 24 h of NMs exposure. We further showed that nano-TiO2 mediated cell death is independent of the classical p53-Bax apoptosis pathway. Our findings provided insights into the potential cellular fates of human oral epithelial cells as they interface with industrial grade nano-HA and nano-TiO2.
Abstract Introduction Complete eradication of microorganisms is essential for successful root canal therapy. However, current methods to evaluate persistent bacteria after therapy are not widely ...practiced. Adenosine triphosphate (ATP) is an indicator of viable cells. The bioluminescence-based ATP assay is easy to perform, and results can be obtained in a clinically relevant time frame of 5 minutes. The aims of this study were to evaluate the sensitivity of the ATP detection method and the specificity of this assay for viable cells and to compare the ATP and culture methods from root canal samples of patients undergoing endodontic treatment. Methods The sensitivity of the ATP assay was determined using bacterial species commonly isolated from root canals. Bacteria were treated with sodium hypochlorite; after which, culture plating and the ATP assay were performed. Forty-three root canal samples before (S1) and after (S2) instrumentation and 36 samples after the removal of calcium hydroxide dressing (S3) were collected from patients undergoing root canal treatment and subjected to ATP assay and anaerobic culture. Results The sensitivity of the ATP assay was determined to be between 10 and 100 bacterial cells. This method of detection also correlated well with the presence of viable bacteria. The ATP readings obtained allowed clear segregation of anaerobic culture-positive and -negative samples obtained from infected root canals of patients. Conclusions The ATP detection method can be used as a rapid tool to determine the presence of viable bacteria during root canal therapy. This method may be potentially useful as an adjunct to root canal treatment.
Individuals with type 2 diabetes are at increased risk of acquiring melioidosis, a disease caused by Burkholderia pseudomallei infection. Although up to half of melioidosis patients have underlying ...diabetes, the mechanisms involved in this increased susceptibility are unknown. We found that B. pseudomallei-infected PBMCs from diabetic patients were impaired in IL-12p70 production, which resulted in decreased IFN-γ induction and poor bacterial killing. The defect was specific to the IL-12-IFN-γ axis. Defective IL-12 production was also observed during Mycobacterium tuberculosis infection, in which diabetes is likewise known to be a strong risk factor. In contrast, IL-12 production in diabetic cells was not affected upon Salmonella enterica infection or in response to TLR2, -3, -4, and -5 ligands. Poor IL-12 production correlated with a deficiency in intracellular reduced glutathione (GSH) concentrations in diabetic patients. Addition of GSH or N-acetylcysteine to PBMCs selectively restored IL-12 and IFN-γ production and improved bacterial killing. Furthermore, the depletion of GSH in mice led to increased susceptibility to melioidosis, reduced production of IL-12p70, and poorer disease outcome. Our data thus establish a link between GSH deficiency in diabetes and increased susceptibility to melioidosis that may open up new therapeutic avenues to protect diabetic patients against some intracellular bacterial pathogens.
Abstract Introduction Successful endodontic treatment outcomes require new strategies for the complete eradication of microbial biofilms in the root canal system. Matrix metalloproteinases (MMPs) are ...essential enzymes in microbial cell growth and homeostasis, and they require transition metal ion cofactors to function. Targeting MMP activity also preserves dentin collagen integrity. In this study, 1,10-phenanthroline-5,6-dione (Phendione), a metal chelator, was tested as a potentially novel antimicrobial agent against Enterococcus faecalis and inhibitor of human MMP in the root canal. Methods Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of Phendione on E. faecalis were determined. The antimicrobial properties of Phendione in the presence of dentin powder and various transition metal ions were examined. The ability of Phendione to inhibit human MMP-2 was subsequently tested. The efficacy of Phendione against E. faecalis biofilm was determined by exposure of 7-day-old E. faecalis biofilms to Phendione. Results The MIC and MBC of Phendione were 2.0 μg/mL and 16 μg/mL, respectively, whereas 64 μg/mL was required to kill E. faecalis biofilm. Phendione completely eradicated E. faecalis despite dentin preincubation. The presence of Zn2+ , and to a lesser extent Fe2+ , abrogated the antimicrobial effect of Phendione. In addition, Phendione at MIC and MBC significantly inhibited human MMP-2 activity. Conclusions Phendione effectively eradicated E. faecalis biofilms and significantly inhibited human MMP-2 through its ability to chelate metal ions. The antibacterial property of Phendione was preserved in the presence of dentin. Phendione can potentially be applied in endodontic treatment as both an antimicrobial agent and MMP inhibitor.
There is little knowledge about factors which may affect oral health among older adults. The objective of this study was to determine the relationship between Streptococcus mutans (MS) and ...Lactobacilli (LB) counts and caries among older adults.
In this community-based observation study, 141 participants aged 60 years and above were recruited from the west district of Singapore. Alongside the clinical examination, saliva samples were collected to determine Streptococcus mutans (MS) and Lactobacilli (LB) counts, as well as to record salivary flow rate and buffering capacity of saliva.
Of the 141 participants, 63.8% were female and 94.3% were of Chinese ethnicity. The mean DMFT was 11.08 (s.d. 8.27). 9.9% of participants had at least one decayed tooth, 52.5% had minimum one missing tooth and 86.5% had at least one filled tooth. 67.4% had MS counts of ≥ 10
while LB counts were ≥ 10
for 48.2%. 83.7% had normal salivary flow or hypersalivation (> = 1 mL/min), the buffering capacity of the saliva was alkaline in 61% of the participants. Multivariate analysis showed that participants who had high MS counts were less likely to have a DMFT < 12 OR (95% CI), 0.29 (0.11-0.77) whereas participants who had high LB counts were less likely to have a DMFT ≤ 14 OR (95% CI), 0.45 (0.20-1.002).
Our study showed a positive correlation between MS and LB counts and caries experience in older adults. The mean DMFT was on the low side in our sample despite having a relatively high MS count. This suggests that there are many other factors which vary according to host environment, physiological and biological conditions that may affect MS and LB counts in the oral cavity.
Our study supports the knowledge that the aetiology of dental caries among older adults is a complex process and it would be wrong to consider caries as a same problem with the same solution for all age groups.
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