Synthesis and antimicrobial activities of N6-hydroxyagelasine analogs and revision of the structure of ageloximes.

(+)-N6-Hydroxyagelasine D, the enantiomer of the proposed structure of (-)-ageloxime D, as well as N6-hydroxyagelasine analogs were synthesized by selective N-7 alkylation of N6-[tert-butyl(dimethyl)silyloxy]-9-methyl-9H-purin-6-amine in order to install the terpenoid side chain, followed by fluoride mediated removal of the TBDMS-protecting group. N6-Hydroxyagelasine D and the analog carrying a geranylgeranyl side chain displayed profound antimicrobial activities against several pathogenic bacteria and protozoa and inhibited bacterial biofilm formation. However these compounds were also toxic towards mammalian fibroblast cells (MRC-5). The spectral data of N6-hydroxyagelasine D did not match those reported for ageloxime D before. Hence, a revised structure of ageloxime D was proposed. Basic hydrolysis of agelasine D gave (+)-N-[4-amino-6-(methylamino)pyrimidin-5-yl]-N-copalylformamide, a compound with spectral data in full agreement with those reported for (-)-ageloxime D.

Environmental fate and effects of novel quorum sensing inhibitors that can control biofilm formation.

The formation of bacterial biofilms can have negative impacts on industrial processes and are typically difficult to control. The increase of antibiotic resistance, in combination with the requirement for more environmentally focused approaches, has placed pressure on industry and the scientific community to reassess biofilm control strategies. The discovery of bacterial quorum sensing, as an important mechanism in biofilm formation, has led to the development of new substances (such as halogenated thiophenones) to inhibit the quorum sensing process. However, there are currently limited data regarding the biodegradability or ecotoxicity of these substances. To assess the environmental fate and effects of thiophenones capable of quorum sensing inhibition, candidate substances were first identified that have potentially high biodegradability and low ecotoxicity using quantitative structure activity relationships. Subsequent confirmatory hazard assessment of these substances, using a marine alga and a marine crustacean, indicated that these estimates were significantly under predicted with acute toxicity values more than three orders of magnitude lower than anticipated combined with limited biodegradability. Therefore, although these quorum sensing inhibitors appear a promising approach to control biofilms, they may also have environmental impacts on certain aquatic organisms.

Specific quorum sensing-disrupting activity (A QSI) of thiophenones and their therapeutic potential.

Disease caused by antibiotic resistant pathogens is becoming a serious problem, both in human and veterinary medicine. The inhibition of quorum sensing, bacterial cell-to-cell communication, is a promising alternative strategy to control disease. In this study, we determined the quorum sensing-disrupting activity of 20 thiophenones towards the quorum sensing model bacterium V. harveyi. In order to exclude false positives, we propose a new parameter (AQSI) to describe specific quorum sensing activity. AQSI is defined as the ratio between inhibition of quorum sensing-regulated activity in a reporter strain and inhibition of the same activity when it is independent of quorum sensing. Calculation of AQSI allowed to exclude five false positives, whereas the six most active thiophenones (TF203, TF307, TF319, TF339, TF342 and TF403) inhibited quorum sensing at 0.25 µM, with AQSI higher than 10. Further, we determined the protective effect and toxicity of the thiophenones in a highly controlled gnotobiotic model system with brine shrimp larvae. There was a strong positive correlation between the specific quorum sensing-disrupting activity of the thiophenones and the protection of brine shrimp larvae against pathogenic V. harveyi. Four of the most active quorum sensing-disrupting thiophenones (TF 203, TF319, TF339 and TF342) were considered to be promising since they have a therapeutic potential of at least 10.

The effect of metronidazole on the presence of P. gingivalis and T. forsythia at 3 and 12 months after different periodontal treatment strategies evaluated in a randomized, clinical trial.

OBJECTIVE: The benefit of full-mouth disinfection (FDIS) over traditional scaling and root planing (SRP) in the treatment of chronic, destructive periodontitis remains equivocal and it is not known whether the use of adjunctive antibiotics may enhance the effect of FDIS. Therefore, the aim of this study was to evaluate the effect of conventional SRP completed over 21 days or 1-day FDIS, with or without systemically delivered adjunctive metronidazole (MET) on the presence of P. gingivalis and T. forsythia after 3 and 12 months. MATERIALS AND METHODS: One hundred and eighty-four patients with moderate-to-severe periodontitis were randomly allocated to one of four treatment groups; (1) FDIS+MET; (2) FDIS+placebo; (3) SRP+MET; (4) SRP+placebo. Prior to treatment, pooled subgingival samples were obtained from the five deepest pockets. The same sites were sampled again 3 and 12 months after treatment. All samples were analyzed for P. gingivalis and T. forsythia by PCR, whereas A. actinomycetemcomitans and other bacteria were identified by culture techniques. RESULTS: At baseline, 47% of the samples were positive for P. gingivalis, while almost all samples were positive for T. forsythia. The occurrence of P. gingivalis and T. forsythia was significantly reduced at 3 and 12 months after treatment in the FDIS+MET group, but not in the other treatment groups. CONCLUSION: FDIS+MET had a significant effect in patients with P. gingivalis and T. forsythia, resulting in a significant reduction in number of patients where these micro-organisms could be detected at 3 and 12 months post-therapy.

A quorum sensing-disrupting brominated thiophenone with a promising therapeutic potential to treat luminescent vibriosis.

Vibrio harveyi is amongst the most important bacterial pathogens in aquaculture. Novel methods to control this pathogen are needed since many strains have acquired resistance to antibiotics. We previously showed that quorum sensing-disrupting furanones are able to protect brine shrimp larvae against vibriosis. However, a major problem of these compounds is that they are toxic toward higher organisms and therefore, they are not safe to be used in aquaculture. The synthesis of brominated thiophenones, sulphur analogues of the quorum sensing-disrupting furanones, has recently been reported. In the present study, we report that these compounds block quorum sensing in V. harveyi at concentrations in the low micromolar range. Bioluminescence experiments with V. harveyi quorum sensing mutants and a fluorescence anisotropy assay indicated that the compounds disrupt quorum sensing in this bacterium by decreasing the ability of the quorum sensing master regulator LuxR to bind to its target promoter DNA. In vivo challenge tests with gnotobiotic brine shrimp larvae showed that thiophenone compound TF310, (Z)-4-((5-(bromomethylene)-2-oxo-2,5-dihydrothiophen-3-yl)methoxy)-4-oxobutanoic acid, completely protected the larvae from V. harveyi BB120 when dosed to the culture water at 2.5 µM or more, whereas severe toxicity was only observed at 250 µM. This makes TF310 showing the highest therapeutic index of all quorum sensing-disrupting compounds tested thus far in our brine shrimp model system.

Thiophenones inhibit Staphylococcus epidermidis biofilm formation at nontoxic concentrations.

Frequent use of medical implants has led Staphylococcus epidermidis to develop into an opportunistic pathogen. The virulence is mainly linked to biofilm formation. Infections associated with biofilms are difficult to treat owing to enhanced resistance to antibiotics. Therefore, new and alternative treatments are called for. Bacterial communication is one of the regulatory mechanisms suggested to be involved in coordinating biofilm formation. In this study, we compared three communication inhibitors for preventing in vitro biofilm formation: a synthetic furanone, and two synthetic thiophenones, which are sulphur analogues of furanones. Furanones naturally source from the red macro alga Delisea pulchra. We also investigated the effect of thiophenone on transcriptional levels of genes associated with biofilm formation. We found that thiophenones were more effective in inhibiting biofilm formation than furanone, also in presence of albumin. We furthermore found that the thiophenones inhibited biofilm formation and bacterial communication more than furanones, and were less cytotoxic. The expression of the icaC and the lrgB genes, which are associated with biofilm formation, were affected by the thiophenone.

The association between selected risk indicators and severity of peri-implantitis using mixed model analyses.

AIM: The aim of the study was to assess possible risk indicators for peri-implantitis at different levels of severity using multi-level analyses. MATERIAL AND METHODS: One hundred and nine subjects attended the examination, 69 females and 40 males. Mean time of implants in function was 8.4 years (standard deviation 4.6) (subject level). The participants were examined clinically and radiographically. Information regarding general health and habits was gathered, with special emphasis on smoking, oral hygiene and susceptibility to periodontitis. The relation between possible risk indicators and the following features were assessed: • Detectable peri-implantitis: detectable radiographic bone loss (>0.4 mm) and inflammation • Overt peri-implantitis: radiographic peri-implant bone loss 2.0 mm and bleeding on probing /suppuration at pocket probing depth 4 mm. RESULTS: Multi-level statistical analyses identified location in the maxilla as risk indicator for detectable peri-implantitis. Regarding overt peri-implantitis, gender (male) and history of periodontitis were identified as risk indicators. CONCLUSION: Individuals with a history of periodontitis were prone to peri-implantitis, peri-implant bone loss ≥ 2.0 mm and overt in the present study. No association was found between smoking and peri-implant disease in the present study population.

Natural transformation of oral streptococci.

Natural transformation is found in most groups of oral streptococci, including the mitis, the anginosus, and the mutans groups. This ability has been applied as a powerful tool to explore streptococcal gene functions and regulatory pathways, particularly in Streptococcus mutans and Streptococcus gordonii. The range of strains and species amenable to transformation has expanded in recent years with the identification of several competence-stimulating peptide signals (CSPs). In this chapter we present protocols for natural transformation in strains found in the three groups of transformable oral streptococci, with focus on methods using synthetic CSPs. We also include suggestions on how to optimize competence conditions for individual species or strains.

Does the wide use of quaternary ammonium compounds enhance the selection and spread of antimicrobial resistance and thus threaten our health?

Quaternary ammonium compounds (QACs) are widely used biocides that possess antimicrobial effect against a broad range of microorganisms. These compounds are used for numerous industrial purposes, water treatment, antifungal treatment in horticulture, as well as in pharmaceutical and everyday consumer products as preserving agents, foam boosters, and detergents. Resistance toward QACs is widespread among a diverse range of microorganisms and is facilitated by several mechanisms such as modifications in the membrane composition, expression of stress response and repair systems, or expression of efflux pump genes. Development of resistance in both pathogenic and nonpathogenic bacteria has been related to application in human medicine and the food industry. QACs in cosmetic products will inevitably come into intimate contact with the skin or mucosal linings in the mouth and thus are likely to add to the selection pressure toward more QAC-resistant microorganisms among the skin or mouth flora. There is increasing evidence of coresistance and cross-resistance between QACs and a range of other clinically important antibiotics and disinfectants. Use of QACs may have driven the fixation and spread of certain resistance cassette collectors (class 1 integrons), currently responsible for a major part of antimicrobial resistance in gram-negative bacteria. More indiscriminate use of QACs such as in cosmetic products may drive the selection of further new genetic elements that will aid in the persistence and spread of antimicrobial resistance and thus in limiting our treatment options for microbial infections.

Prevalence of peri-implantitis related to severity of the disease with different degrees of bone loss.

BACKGROUND: Several measurements are combined to diagnose peri-implant disease, and different thresholds are used to describe the disease. The purpose of this study was to evaluate the prevalence of peri-implant disease and to apply different diagnostic thresholds to assess its prevalence in relation to severities of peri-implantitis with different degrees of bone loss. METHODS: A total of 164 subjects with dental implants inserted at the Institute of Clinical Odontology, University of Oslo, between 1990 and 2005, were invited to join the project, and 109 subjects attended the examination (mean age: 43.8 years; range: 18 to 80 years). The mean functional loading time was 8.4 years (SD: 4.6 years). The participants were examined clinically and radiographically. The following aspects of disease were assessed to describe the peri-implant condition: detectable radiographic peri-implant bone loss and inflammation, the presence of bleeding on probing at a probing depth >or=4 or >or=6 mm, and radiographic peri-implant bone loss assessed at >or=2.0 and >or=3.0 mm. RESULTS: Assessing peri-implantitis at different levels of severity yielded a substantial variance in prevalence (11.3% to 47.1%) in the present study population. CONCLUSION: Peri-implant inflammation was a frequent finding with and without peri-implant bone loss.

Prevalence of implant loss and the influence of associated factors.

BACKGROUND: The objective of this study was to assess the outcome of dental implants inserted at the Institute of Clinical Odontology, University of Oslo, between 1990 and 2005. The prevalence of implant loss and the factors associated with the outcome were studied. METHODS: A total of 164 subjects were invited to participate in this cross-sectional project, of whom 55 were unable, leaving 109 volunteers available for examination. The study population included 69 females and 40 males with a mean age of 43.8 years at the time of implant insertion (range, 18 to 80 years). At the subject level, the mean time from implant loading to the present examination was 8.4 years (range, 1.1 to 16.0 years). The participants were examined clinically and radiographically and interviewed regarding general health and habits. RESULTS: The 109 examined subjects had been treated with 374 implants. Eighteen implants (4.8%) were lost in 10 subjects (9.2%). Eleven implants were lost before loading, three were lost during the first 5 years after loading, and four were lost 5 to 10 years after loading. No implants were lost after >10 years of loading. The loss of oral implants was significantly associated with a history of smoking and periodontitis (P <0.05). CONCLUSIONS: The inserted implants showed a high survival rate, especially after the first year of insertion, even though the subjects were not maintained by specialists. All late implant losses were preceded by an early loss. Implant loss was significantly associated with smoking and periodontitis.

Furanones, potential agents for preventing Staphylococcus epidermidis biofilm infections?

OBJECTIVES: Staphylococcus epidermidis is often associated with biofilm infections related to medical implants. The aim of the present study was to find furanones that decrease biofilm formation without irritative or genotoxic effects, or effects on S. epidermidis growth. METHODS: After screening including bioluminescence and biofilm assays, 2 furanones out of 11 were chosen for further studies. MIC values of the two furanones were established to determine whether biofilm inhibition effects were ascribed to inhibition of bacterial growth. To further investigate interference with communication, the effect of the furanones was tested in the presence of the autoinducer-2 precursor (S)-4,5-dihydroxy-2,3-pentanedione. The furanones were tested for possible irritative effects by the Hen's egg test chorioallantoic membrane procedure. Finally, potential genotoxic effects in mice were assessed by a membrane array, and effects on global gene expression were investigated by using a microarray representing 30,000 genes of the mouse genome. RESULTS: From the bioluminescence assay, 4 furanones out of 11 were chosen for further biofilm analyses. Biofilm formation by S. epidermidis was significantly decreased by the four furanones tested at concentrations not affecting microbial growth. Two furanones were chosen for further studies: one that decreased biofilm statistically more than the others and one containing two bromo substituents. The two furanones were found to be non-irritative and non-genotoxic at the concentrations used. CONCLUSIONS: Furanones may inhibit biofilm formation through interference with quorum sensing and thus represent promising agents for protecting surfaces from being colonized by S. epidermidis.

AI-2 quorum sensing affects antibiotic susceptibility in Streptococcus anginosus.

OBJECTIVES: The concern over rising antibiotic resistance necessitates exploration of alternative approaches in antimicrobial therapy. Bacterial communities use the auto-inducer 2 (AI-2) quorum sensing signal at a specific threshold level for intra- and interspecies communication in order to regulate virulence behaviour. AI-2 signal production occurs in bacteria that possess a luxS homologue. In this study, we investigate for the first time the association between AI-2 signalling and susceptibility to antibiotics. METHODS: Streptococcus anginosus wild-type and its isogenic luxS mutant SA001 were exposed to erythromycin and ampicillin. Susceptibility to erythromycin and ampicillin was determined by measuring the cell density and viability. Complementation assays were conducted by exposing the mutant to wild-type supernatant or to the AI-2 precursor molecule dihydroxy-2,3-pentanedione (DPD). RESULTS: Disruption of luxS in S. anginosus resulted in a mutant with increased susceptibility to erythromycin and ampicillin. Supernatant from S. anginosus wild-type partially restored growth of SA001 in the presence of the two antibiotics. DPD restored growth of the luxS mutant in the presence of erythromycin and ampicillin to values similar to that of S. anginosus wild-type. CONCLUSIONS: Our results indicate that luxS-based AI-2 communication is associated with antibiotic susceptibility. Targeting the AI-2 signal communication may present a novel approach in antimicrobial therapy.

LuxS-mediated signalling in Streptococcus anginosus and its role in biofilm formation.

The autoinducer-2 signal (AI-2) produced by several Gram-positive and Gram-negative bacteria mediates interspecies communication. In this study we were able to identify an orthologue of luxS, required for the synthesis of AI-2 signals, in Streptococcus anginosus. Comparative analyses revealed conserved sequences in the predicted S. anginosus LuxS. Expression of luxS was highest during early exponential growth phase. Compared to other oral streptococci, conditioned media from growth of members of the anginosus group were the most efficient in inducing bioluminescence in Vibrio harveyi, indicative of AI-2 signalling. Disruption of luxS in S. anginosus resulted in a mutant deficient in biofilm formation, whereas no effect on planktonic growth rate was observed under various growth conditions. S. anginosus is part of the human flora found in biofilms of the oral cavity, as well as of the upper respiratory, gastrointestinal and urogenital tracts. Such habitats harbour large varieties of bacterial species, among which cell-cell communication may play an important role. S. anginosus has also been associated with purulent infections and cancer in the upper digestive tract. Knowledge about the molecular mechanisms involved in S. anginosus communication is important for understanding its commensalism and its pathogenic transition.

Purification and functional studies of a potent modified quorum-sensing peptide and a two-peptide bacteriocin in Streptococcus mutans.

Bacteria use quorum-sensing signals or autoinducers to communicate. The signals in Gram-positive bacteria are often peptides activated by proteolytic removal of an N-terminal leader sequence. While investigating stimulation of antimicrobial peptide production by the Streptococcus mutans synthetic competence stimulating peptide signal (21-CSP), we found a peptide similar to the 21-CSP, but lacking the three C-terminal amino acid residues (18-CSP). The 18-CSP was more potent in inducing competence, biofilm formation, and antimicrobial activity than the 21-CSP. Our results indicate that cleavage of the three C-terminal residues occurred post export, and was not regulated by the CSP-signalling system. Deletion of comD encoding the CSP receptor abolished the competence and biofilm responses to the 21-CSP and the 18-CSP, suggesting that signal transduction via the ComD receptor is involved in the responses to both CSPs. In S. mutans GS5, beside the 18-CSP we also purified to homogeneity a two-peptide bacteriocin which production was stimulated by the 18-CSP and the 21-CSP. Partial sequence of the two-peptide bacteriocin revealed the product of the smbAB genes recently described. We found that the peptide SmbB was slightly different from the deduced sequence, and confirmed the prediction that both peptides constituting SmbAB bacteriocin are post-translationally modified. SmbAB exhibited antimicrobial activity against 11 species of streptococci, Enterococcus faecalis and Staphylocococcus epidermidis. Taken together, the findings support the involvement of the CSP response in bacteriocin production by streptococci and suggest a novel strategy to potentiate autoinducer activity.

THE BIOFILM CONCEPT: CONSEQUENCES FOR FUTURE PROPHYLAXIS OF ORAL DISEASES?

Biofilm control is fundamental to oral health. Existing oral prophylactic measures, however, are insufficient. The main reason is probably because the micro-organisms involved organize into complex biofilm communities with features that differ from those of planktonic cells. Micro-organisms have traditionally been studied in the planktonic state. Conclusions drawn from many of these studies, therefore, need to be revalidated. Recent global approaches to the study of microbial gene expression and regulation in non-oral micro-organisms have shed light on two-component and quorum-sensing systems for the transduction of stimuli that allow for coordinated gene expression. We suggest interference with two-component and quorum-sensing systems as potential novel strategies for the prevention of oral diseases through control of oral biofilms. Information is still lacking, however, on the genetic regulation of oral biofilm formation. A better understanding of these processes is of considerable importance.