Trans-cinnamaldehyde potently kills Enterococcus faecalis biofilm cells and prevents biofilm recovery.

Enterococcus faecalis is a biofilm-forming, nosocomial pathogen that is frequently isolated from failed root canal treatments. Contemporary root canal disinfectants are ineffective in eliminating these biofilms and preventing reinfection. As a result, there is a pressing need to identify novel and safe antibiofilm molecules. The effect of short-term (5 and 15 min) and long-term (24 h) treatments of trans-cinnamaldehyde (TC) on the viability of E. faecalis biofilms was compared with currently used root canal disinfectants. Treatment for 15 min with TC reduced biofilm metabolic activity as effective as 1% sodium hypochlorite and 2% chlorhexidine. Treatment with TC for 24 h was significantly more effective than 2% chlorhexidine in reducing the viable cell counts of biofilms. This serendipitous effect of TC was sustained for 10 days under growth-favoring conditions. For the first time, our study highlights the strong antibacterial activity of TC against E. faecalis biofilms, and notably, its ability to prevent biofilm recovery after treatment.

A Novel Small Molecule, 1,3-di-m-tolyl-urea, Inhibits and Disrupts Multispecies Oral Biofilms.

An imbalance of homeostasis between the microbial communities and the host system leads to dysbiosis in oral micro flora. DMTU (1,3-di-m-tolyl-urea) is a biocompatible compound that was shown to inhibit Streptococcus mutans biofilm by inhibiting its communication system (quorum sensing). Here, we hypothesized that DMTU is able to inhibit multispecies biofilms. We developed a multispecies oral biofilm model, comprising an early colonizer Streptococcus gordonii, a bridge colonizer Fusobacterium nucleatum, and late colonizers Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans. We performed comprehensive investigations to demonstrate the effect of DMTU on planktonic cells and biofilms. Our findings showed that DMTU inhibits and disrupts multispecies biofilms without bactericidal effects. Mechanistic studies revealed a significant down regulation of biofilm and virulence-related genes in P. gingivalis. Taken together, our study highlights the potential of DMTU to inhibit polymicrobial biofilm communities and their virulence.

Secretory products of Escherichia coli biofilm modulate Candida biofilm formation and hyphal development.

AIM: To investigate the time- and concentration-dependent effects of Escherichia coli biofilm supernatant on Candida biofilm development, and to assess the effect of E. coli supernatant on Candida albicans hypha-specific genes (HSGs) expression. METHODS: The effect of E. coli biofilm supernatant on six Candida spp. was assessed by tetrazolium salt (XTT) reduction assay, scanning electron microscopy (SEM), and confocal laser scanning microscopy (CLSM). The effect of biofilm supernatant on the expression of C. albicans HSGs (ECE1, HWP1, HYR1, RBT1, RBT4, ALS3, and ALS8) and transcription factors (CPH1, CPH2, EFG1, TEC1, RAS1, TUP1, NRG1 and RFG1) was evaluated with real-time polymerase chain reaction (PCR). RESULTS: Escherichia coli biofilm secretory products significantly inhibited C. albicans, C. glabrata, C. tropicalis and C. krusei biofilms at 24 h and all Candida spp. at 48 h (P < 0.05), and SEM and CLSM confirmed these data. HSGs RBT1 and RBT4 were mostly up-regulated and ECE1, HWP1 and HYR1 were mostly down-regulated. ALS3 was totally suppressed. All HSGs were down-regulated at 48 h (P < 0.05). NRG1, RFG1 and EFG1, CPH1 and TEC1, and TUP1 and CPH2 showed similar expression trends and all were down-regulated at 48 h (P < 0.05). CONCLUSIONS: Escherichia coli secretory elements significantly impair Candida biofilm development possibly by modulating HSGs and its transcriptional regulation.

Human serum promotes Candida albicans biofilm growth and virulence gene expression on silicone biomaterial.

OBJECTIVES: Systemic candidal infections are a common problem in hospitalized patients due to central venous catheters fabricated using silicone biomaterial (SB). We therefore evaluated the effect of human serum on C. albicans biofilm morphology, growth, and the expression of virulence-related genes on SB in vitro. METHODS: We cultivated C. albicans SC5314 (wild-type strain, WT) and its derivative HLC54 (hyphal mutant, HM) for 48 h in various conditions, including the presence or absence of SB discs, and human serum. The growth of planktonic and biofilm cells of both strains was monitored at three time points by a tetrazolium salt reduction assay and by scanning electron microscopy. We also analyzed by RT-PCR its expression of the virulence-related genes ALS3, HWP1, EAP1, ECE1, SAP1 - SAP10, PLB1, PLB2, PLC and PLD. RESULTS: At each time point, planktonic cells of WT strain cultured in yeast nitrogen base displayed a much higher expression of EAP1 and HWP1, and a moderately higher ALS3 expression, than HM cells. In planktonic cells, expression of the ten SAP genes was higher in the WT strain initially, but were highly expressed in the HM strain by 48 h. Biofilm growth of both strains on SB was promoted in the presence of human serum than in its absence. Significant upregulation of ALS3, HWP1, EAP1, ECE1, SAP1, SAP4, SAP6 - SAP10, PLB1, PLB2 and PLC was observed for WT biofilms grown on serum-treated SB discs for at least one time point, compared with biofilms on serum-free SB discs. CONCLUSIONS: Human serum stimulates C. albicans biofilm growth on SB discs and upregulates the expression of virulence genes, particularly adhesion genes ALS3 and HWP1, and hydrolase-encoding genes SAP, PLB1 and PLB2. This response is likely to promote the colonization of this versatile pathogen within the human host.

Expression and localization of cystic fibrosis transmembrane conductance regulator in human gingiva.

CFTR (cystic fibrosis transmembrane conductance regulator) is a cAMP-activated chloride channel that regulates electrolyte and water transport. The present study investigated the expression and localization of CFTR in human gingiva and explored the possible association of CFTR with periodontal conditions. CFTR expression in gingival biopsies from five periodontally healthy subjects and ten subjects with chronic periodontitis and in the RHGE (reconstituted human gingival epithelia) was detected by immunohistochemistry, whereas its expression in gingival biopsies was analysed by immunofluorescence staining. CFTR mRNA was analysed by reverse transcription-PCR. CFTR mRNA was detected in human gingival epithelia and RHGE. CFTR protein was detected in gingival biopsies from both healthy subjects and individuals with periodontitis and in RHGE. In healthy subjects, CFTR expression was mainly confined to the granular and spinous layers of epithelia and localized on the cell membrane. In patients with periodontitis, CFTR was detected in all layers of epithelia and the underlying connective tissues. The mean CFTR expression levels in periodontitis patients were significantly higher than those in healthy subjects. The present study for the first time showed the expression and localization of CFTR in human gingival epithelia. Elevated CFTR expression in periodontitis subjects implies the possible involvement of CFTR in periodontal disease pathogenesis. Further study is warranted to confirm the present findings.

Characterization of Actinobacillus actinomycetemcomitans isolated from young Chinese aggressive periodontitis patients.

OBJECTIVE: This study characterized Actinobacillus actinomycetemcomitans isolates from young Chinese aggressive periodontitis patients. METHODS: Subgingival plaque samples (two/subject) were collected from diseased subjects < 25 years old (n = 9, mean age 21.1 +/- 1.6 years) and age-matched periodontitis-free controls (n = 47, mean age 22.0 +/- 1.1 years). Selective and anaerobic culture were used. The serotype, leukotoxin gene (ltx) operon promoter and the cytolethal distending toxin (cdt) genes complex of the A. actinomycetemcomitans isolates were investigated. Effects of the isolates on non-keratinizing periodontal ligament epithelial cells monolayer were studied. RESULTS: Diseased subjects had significantly higher full-mouth bleeding score (p = 0.002) and total viable counts from plaque samples (7.2 x 10(6) vs. 2.1 x 10(5) CFU/paperpoint, p < 0.005). A. actinomycetemcomitans was isolated from 67%/56% or 6%/4% of diseased or controls subject/sites, respectively (p < 0.001). The proportion of A. actinomycetemcomitans isolatable from aggressive periodontitis or periodontitis-free associated subgingival plaque was low (0.7% vs. 0.1%, p < 0.02). The serotype of the isolates was characterized. All isolates possessed 652-like ltx gene promoter and all but one serotype c isolate from a diseased patient had intact cdtABC genes. That particular strain appeared to confer the least cellular damages on periodontal ligament epithelial monolayer compared to others. CONCLUSION: This preliminary study confirmed the notion of increased prevalence and quantity of A. actinomycetemcomitans associated with aggressive periodontitis in young patients. The overall ltx promoter and cdt characteristics of the A. actinomycetemcomitans isolates, however, were similar among the diseased and control groups. A strain lacking the cdtABC gene appeared to be less damaging to a periodontal ligament epithelial cell model. Further studies therefore are warranted to clarify the pathogenic role and potentials of A. actinomycetemcomitans in aggressive periodontitis.

Direct detection of Actinomyces spp. from infected root canals in a Chinese population: a study using PCR-based, oligonucleotide-DNA hybridization technique.

OBJECTIVES: The poor sensitivity of phenotypic identification techniques has hampered the taxonomic differentiation of Actinomyces. Hence we developed a sensitive and specific, PCR-based oligonucleotide-DNA hybridization technique to detect Actinomyces spp. and, used this method to detect these organisms in samples directly obtained from infected root canals. METHODS: A total of 32 samples from 28 Chinese patients, with primary root canal infections, aseptically exposed at the first patient visit, were studied. Whole bacterial genomic DNA was isolated directly from paper point samples. The variable regions of 16S ribosomal DNA of bacteria were amplified and labeled with digoxigenin for further hybridization and detection. A total of seven oligonucleotide probes specific for A. bovis, A. gerencseriae, A. israelii, A. meyeri, catalase-negative A. naeslundii (genospecies 1 and 2), catalase-positive A. naeslundii genospecies 2 and A. odontolyticus were used. RESULTS: 16 of the 32 teeth were infected with one or more Actinomyces species. The prevalence rates of the examined species were: A. odontolyticus 31.3%, A. meyeri 9.4%, A. naeslundii 9.4%, A. israelii 6.3% and A. gerencseriae 3.1%; no A. bovis was detected in any of the canals. Furthermore, A. odontolyticus was isolated more frequently from root canals with caries or a history of caries (Fisher's exact test: P=0.0496; Odds ratio=9.00, 95% confidence interval: 0.97-83.63), and A. naeslundii was significantly associated with traumatized teeth (Fisher's exact test: P=0.0121; Odds ratio=57.00, 95% confidence interval: 2.10-1546.90). However, no significant correlation was found between Actinomyces spp. and clinical symptoms and signs, such as pain, swelling, percussion to tenderness, sinus and periapical radiolucency. CONCLUSION: Actinomyces spp. may be important pathogens of root canal infections. A. naeslundii in particular may be related with traumatized teeth. A. odontolyticus appears to be involved in infections related to caries, exposure of dentinal tubules during cavity preparation and/or leaking restoration, but further clarification with large samples is necessary.