Amino acid substitutions in specific proteins correlate with farnesol unresponsiveness in Candida albicans.

BACKGROUND: The quorum-sensing molecule farnesol, in opportunistic yeast Candida albicans, modulates its dimorphic switch between yeast and hyphal forms, and biofilm formation. Although there is an increasing interest in farnesol as a potential antifungal drug, the molecular mechanism by which C. albicans responds to this molecule is still not fully understood. RESULTS: A comparative genomic analysis between C. albicans strains that are naturally unresponsive to 30 µM of farnesol on TYE plates at 37 °C versus responsive strains uncovered new molecular determinants involved in the response to farnesol. While no signature gene was identified, amino acid changes in specific proteins were shown to correlate with the unresponsiveness to farnesol, particularly with substitutions in proteins known to be involved in the farnesol response. Although amino acid changes occur primarily in disordered regions of proteins, some amino acid changes were also found in known domains. Finally, the genomic investigation of intermediate-response strains showed that the non-response to farnesol occurs gradually following the successive accumulation of amino acid changes at specific positions. CONCLUSION: It is known that large genomic changes, such as recombinations and gene flow (losses and gains), can cause major phenotypic changes in pathogens. However, it is still not well known or documented how more subtle changes, such as amino acid substitutions, play a role in the adaptation of pathogens. The present study shows that amino acid changes can modulate C. albicans yeast's response to farnesol. This study also improves our understanding of the network of proteins involved in the response to farnesol, and of the involvement of amino acid substitutions in cellular behavior.

Selective bacterial degradation of the extracellular matrix attaching the gingiva to the tooth.

The junctional epithelium (JE) is a specialized portion of the gingiva that seals off the tooth-supporting tissues from the oral environment. This relationship is achieved via a unique adhesive extracellular matrix that is, in fact, a specialized basal lamina (sBL). Three unique proteins - amelotin (AMTN), odontogenic ameloblast-associated (ODAM), and secretory calcium-binding phosphoprotein proline-glutamine rich 1 (SCPPPQ1) - together with laminin-332 structure the supramolecular organization of this sBL and determine its adhesive capacity. Despite the constant challenge of the JE by the oral microbiome, little is known of the susceptibility of the sBL to bacterial degradation. Assays with trypsin-like proteases, as well as incubation with Porphyromonas gingivalis, Prevotella intermedia, and Treponema denticola, revealed that all constituents, except SCPPPQ1, were rapidly degraded. Porphyromonas gingivalis was also shown to alter the supramolecular network of reconstituted and native sBLs. These results provide evidence that proteolytic enzymes and selected gram-negative periodontopathogenic bacteria can attack this adhesive extracellular matrix, intimating that its degradation could contribute to progression of periodontal diseases.

Unexpected diversity in the mobilome of a Pseudomonas aeruginosa strain isolated from a dental unit waterline revealed by SMRT Sequencing.

The Gram-negative bacterium Pseudomonas aeruginosa is found in several habitats, both natural and human-made, and is particularly known for its recurrent presence as a pathogen in the lungs of patients suffering from cystic fibrosis, a genetic disease. Given its clinical importance, several major studies have investigated the genomic adaptation of P. aeruginosa in lungs and its transition as acute infections become chronic. However, our knowledge about the diversity and adaptation of the P. aeruginosa genome to non-clinical environments is still fragmentary, in part due to the lack of accurate reference genomes of strains from the numerous environments colonized by the bacterium. Here, we used PacBio long-read technology to sequence the genome of PPF-1, a strain of P. aeruginosa isolated from a dental unit waterline. Generating this closed genome was an opportunity to investigate genomic features that are difficult to accurately study in a draft genome (contigs state). It was possible to shed light on putative genomic islands, some shared with other reference genomes, new prophages, and the complete content of insertion sequences. In addition, four different group II introns were also found, including two characterized here and not listed in the specialized group II intron database.

Effectiveness of an alcohol-free essential oil-containing mouthwash in institutionalised elders receiving long-term care: a feasibility study.

OBJECTIVES: To conduct a feasibility study on investigating the effectiveness of an alcohol-free essential oil mouthwash (AF-EOMW) to reduce plaque accumulation and oral pathogen levels in institutionalised elders receiving long-term care and to obtain preliminary results. BACKGROUND: Although simple, cost-effective strategies to improve oral hygiene in seniors such as the use of mouthwashes have been shown to reduce the risks of respiratory diseases, little information is available on the feasibility of implementing these measures. METHODS: Twenty-five elderly participants with significant loss of autonomy were initially recruited and divided into two groups. A test group rinsed with an AF-EOMW twice a day, and a control group rinsed with tap water. Data on demographic characteristics, dental history and tobacco use were collected from a questionnaire. Problems encountered during recruitment and data collection were documented. Plaque index, denture cleanliness and salivary levels of several pathogens were measured at three time points: baseline (T0 ), day 22 (T1 ) and day 45 (T2 ). RESULTS: Eighteen participants completed the study. Several problems were encountered during recruitment and execution of the study protocol. No significant differences in clinical or microbiological measures were found between the test group and controls at three time points (p > 0.05). CONCLUSION: This pilot study shows that, if sufficient logistical and financial resources are available, it is feasible to conduct randomised clinical trials in a seniors' facility. The use of an AF-EOMW to improve oral hygiene in seniors was not found to be superior to tap water. However, larger controlled clinical studies are needed to confirm these results.

Interactions between non-phospholipid liposomes containing cetylpyridinium chloride and biofilms of Streptococcus mutans: modulation of the adhesion and of the biodistribution.

Cetylpyridinium chloride (CPC) is a surfactant that binds strongly to bacteria and bacterial biofilms. In this study, fluorescence-based techniques were used to determine the penetration and adhesion of CPC when it was introduced in liposomes. In spite of a reduced adhesion as compared to pure CPC micelles, CPC-containing liposomes adhered significantly to the biofilms of Streptococcus mutans. In contrast, no binding was observed for liposomes that were composed of phosphatidylcholine-cholesterol. The influence of the charge of the liposome on its adhesion to biofilms was studied using cholesterol (Chol) and cholesterol sulfate (Schol). In spite of similar binding to the biofilms, positively charged CPC/Chol liposomes were located mainly in the core of the biofilm microcolonies, whereas the negatively charged CPC/Schol liposomes were mainly concentrated at their periphery. This effect may be attributed to the different availability of the CPC head group. In summary, this work demonstrates the high potential for tailoring drug nanovectors by modulating sterol selection in order to selectively target and bind biofilms.

Palatal brushing for the treatment of denture stomatitis: A multicentre randomized controlled trial.

PURPOSE: To determine the effectiveness of palatal brushing in the treatment of denture-related erythematous stomatitis (DES) in complete denture wearers. METHODS: This two-parallel-arm RCT was conducted in three university clinics in Brazil, Canada, and Chile. Participants (n=77) were randomly allocated to receive (i) instructions for palatal brushing and standard oral/denture hygiene ("intervention"); or (ii) standard oral/denture hygiene instructions only ("control"). Data collection was carried out at the baseline and at 3 and 6 months after intervention. Outcomes included the magnitude of oral Candida carriage and the degree of inflammation of denture-bearing tissues. Groups were compared using generalized estimating equations and chi-square test (α=0.05). RESULTS: Palatal inflammation levels were reduced significantly in the "intervention" compared to "control" group at 6 months (intervention: 70%, control: 40%; chi-square, p=0.04). There was no between-group significant difference in the Candida count from denture and palatal biofilms; however, a subgroup analysis restricted to baseline Candida carriers showed further reduction with the intervention at 6 months. No adversity was observed by trialist or reported by participants. CONCLUSIONS: Including palatal brushing in oral instructions for denture wearers has positive impact on DES-related mucosal inflammation. Thus, our findings endorse the inclusion of palatal brushing in standard oral hygiene instructions to treat DES.

Polymerase chain reaction detection of potentially pathogenic free-living amoebae in dental units.

Several genera of amoebae can be found in water from dental units and on the inner surface of waterlines. The presence of bacterial biofilms on these surfaces is thought to favor the proliferation of amoebae. Potentially pathogenic Acanthamoeba and Naegleria spp. may be an infection risk for patients through contact with open surgical sites or aerosolization. A polymerase chain reaction of DNA extracted from pelleted samples showed that Acanthamoeba spp. and Naegleria spp. were present in water from dental units, suction lines, and suction filters at the dental clinic of the Université de Montréal. Acanthamoeba spp. were detected in 24.2% of 66 samples and Naegleria spp. in 3.0%. We discuss the infection risk associated with these results.

Measurement of airborne bacteria and endotoxin generated during dental cleaning.

Dynamic dental instruments generate abundant aerosols in the work environment. Dental unit waterlines (DUWL) support a large microbial population and can be a significant source of bioaerosols generated during dental treatments. This study was conducted to characterize bioaerosol generation during dental treatments performed in standardized conditions. Culture-based method (R2A, and blood agar with and without O2) and fluorescence microscopy were used. Dental cleaning procedures were performed in an isolated treatment room with controlled ventilation rate. Andersen microbial samplers were used to collect culturable bioaerosols generated before (baseline), during, and after 2 hr of dental treatments. Inhalable dust samplers were used to measure total bioaerosols content in dental hygienist's and patients' breathing zones. AGI-30 were used for the collection of the endotoxin. The use of fluorescence microscopy in combination with culture demonstrated that dental staff and patients were exposed to up to 1.86 E+05 bacteria/m(3) generated during treatments. Fortunately, bioaerosols returned to baseline within 2 hr after the dental procedures. The small diameter of the aerosols generated (< 1 microm) suggests that the risk of contact between the aerosolized bacteria and the respiratory system of exposed individuals is likely to occur.

A new approach to study attached biofilms and floating communities from Pseudomonas aeruginosa strains of various origins reveals diverse effects of divalent ions.

Pseudomonas aeruginosa is an opportunistic pathogen associated with nosocomial infections and disease complications. In the lungs of cystic fibrosis (CF) individuals, biofilm growth plays a crucial role in the persistence and antibiotic resistance of P. aeruginosa. Some strains, adapted to the CF lung microenvironment, show distinguishable phenotypes linked to biofilm production when compared to other strains. Using a novel image analysis quantification approach with crystal violet-stained biofilms, we compared the biofilm formation of four different P. aeruginosa isolates in 24-well plates: PAO1, the reference strain, LESB58 from CF patients' lungs and PPF-1 and Urg-7, two environmental isolates from dental unit waterlines. We also observed the formation of biofilm-like structures (BLSs) floating in the medium and investigated growth inhibition of the attached biofilm and BLS with Mg2+ or Zn2+. Urg-7 produced the most attached biofilms, but not the most BLSs. Attached biofilms had different responses to cations than BLSs did, but the effect of the cations was similar for all strains. These results demonstrate some diversity of biofilm formation in P. aeruginosa and indicate that chemical inhibition of attached biofilm formation for a specific strain or isolate cannot be predicative of a result on other P. aeruginosa strains or on BLSs.

Lawsuit against a dentist related to serious ocular infection possibly linked to water from a dental handpiece.

This case report highlights the risks that may be associated with amoebae in the water of a dental unit. A woman with contact lenses visited her dentist for replacement of a bridge. During the treatment, a stream of water was directed from the handpiece into her right eye. Because of subsequent pain in the eye, the patient consulted several ophthalmologists, who discovered abrasive lesions of the cornea and inflammation. Despite antibacterial and anti-inflammatory treatments, the patient"s visual acuity declined gradually over a period of several days. A microbiological examination nearly 2 months later revealed amoebae in corneal samples. A lawsuit against the dentist was initiated. Although a causal relation with the dental treatment was rejected by the judge in this case, high numbers of amoebae in the water of dental units can present a risk if a patient with pre-existing corneal lesions is splashed. According to the precautionary principle, complete evidence of risk does not have to exist to institute measures to protect individuals and society from that risk. This case reinforces the importance of having patients wear safety glasses during dental treatments and of dental personnel draining the waterlines of dental units, as recommended by the Canadian Dental Association.

Genomic characterisation of environmental Pseudomonas aeruginosa isolated from dental unit waterlines revealed the insertion sequence ISPa11 as a chaotropic element.

The bacterium Pseudomonas aeruginosa is well known to have a remarkable adaptive capacity allowing it to colonise many environments. A recent study on environmental isolates from dental unit waterlines (DUWLs) hinted at a genetic clustering into two groups. Isolates from one of these groups, named cluster III, were shown to have unusual phenotypes for environmental isolates, such as an increased biofilm production. To have a better ecological view, more specifically on isolates from cluster III, the complete genomes of 39 isolates including 16 from DUWLs were sequenced. In addition to an investigation of antibiotic resistance and secretion system gene content, a molecular phylogeny allowed confirmation of the split of the 16 environmental isolates in two groups and also sheds light on a correlation between the phylogenetic positions and the serotypes of the isolates. Isolates from cluster III harboured insertion sequences ISPa11 inserted into the O-specific antigen biosynthesis clusters and the gene lasR, encoding for a master regulator of the quorum sensing. Investigation of key regulators revealed another truncated gene, gacS. Alteration in lasR and gacS genes was consistent with phenotypic assays confirming their inactivation. These results bring new perspectives regarding the ecological adaptive potential of P. aeruginosa.

Improving practice guidelines for the treatment of denture-related erythematous stomatitis: a study protocol for a randomized controlled trial.

BACKGROUND: Denture-related erythematous stomatitis (DES) is a chronic biofilm-mediated disease, affecting one in every three complete denture wearers. Antifungals are the treatment most commonly prescribed by oral health professionals, based on the belief that colonization by Candida spp. is the main cause of DES. However, high recurrence rates and adverse effects are commonly observed, prompting the need for practice guidelines regarding treatment. Results from our pilot study demonstrate that palatal brushing can reduce the palatal inflammation and potentially associated Candida carriage without any need for antifungal therapy. The objective of this study is to validate these pilot results by means of a randomized controlled trial (RCT) and provide a practice guideline for clinicians. METHODS/DESIGN: A pragmatic, two-parallel-arm, multicenter RCT will be conducted in Canada, Brazil, and Chile. Fifty-two adult complete denture wearers presenting with moderate to severe DES will be allocated randomly to two groups: the Intervention arm will consist of palatal brushing and standard oral and denture hygiene measures, while the Control arm will include only standard oral and denture hygiene measures. The study outcome will be the oral Candida carriage. Participants will be assessed at baseline, and at 3 and 6 months post intervention. Descriptive, bivariate, and mixed models with repeated measures will be performed following the intention-to-treat principle. DISCUSSION: This pragmatic RCT will serve to provide a clinical practice guideline regarding the use of preventive measures in the treatment of biofilm-mediated oral diseases. Moreover, it will have a great impact on reducing the harm of antifungal overtreatment on patients suffering from DES. TRIAL REGISTRATION: ClinicalTrials.gov, NCT02686632 . Registered on 15 February 2016.

Biofilm formation by Staphylococcus aureus and Pseudomonas aeruginosa is associated with an unfavorable evolution after surgery for chronic sinusitis and nasal polyposis.

OBJECTIVES: To determine whether biofilm-forming capacity of bacteria demonstrated in chronic rhinosinusitis (CRS) has an impact on persistence of the disease following endoscopic sinus surgery (ESS). METHOD: Thirty-one bacterial strains recovered from 19 patients with CRS at least 1 year post-ESS. Evolution of disease was assessed by questionnaire and endoscopy as favorable or unfavorable. The bacteria were cultured on a 96-well culture plaque and a semi-quantitative method using crystal violet to quantify biofilm production was used. RESULTS: Twenty-two of 31 samples produced a biofilm thicker or equal to the positive control. Biofilm production was noted in 6/10 Pseudomonas aeruginosa isolates, 8/10 Staphylococcus aureus, and 8/11 coagulase-negative staphylococci. Biofilm formation was associated with a poor evolution for Pseudomonas aeruginosa and Staphylococcus aureus, but not coagulase-negative staphylococcus. CONCLUSION: There is a correlation between in vitro biofilm-producing capacity by Pseudomonas aeruginosa and Staphylococcus aureus and unfavorable evolution after ESS, suggesting a role for biofilm production in chronic sinusitis. EBM RATING: B-2b.

Permeability and thermodynamics study of quaternary ammonium surfactants-phosphocholine vesicle system.

Quaternary ammonium compounds (QACs) are recognized as membrane active agents widely used as biocides. The main purpose of this work was to investigate the influence of the QAC head group and acyl chain length on their permeability-perturbing power and on their affinity for lipidic membranes. Permeability perturbations were assessed by measuring the release of calcein entrapped inside vesicles. The affinity of QACs for bilayers was investigated by isothermal titration calorimetry (ITC). QACs bearing C(16) chain were found to be more efficient to decrease the membrane permeability than their C(12) analogues. On the other hand, the chemical nature of the ammonium head group has practically no influence on the permeability perturbations caused by QACs bearing C(16) chains. It was difficult to assess the partitioning of the QACs between the aqueous and lipid phases since the ITC signals could also be associated to morphological changes such as vesicle aggregation. For the systems for which reliable thermodynamic parameters could be obtained, the Gibbs energy of transfer was similar to that for the micellization. The entropy variation represented the main contribution to the Gibbs energy, indicating that the insertion of QACs inside lipidic bilayers is driven by hydrophobic interactions.

Clinical utilization of genomics data produced by the international Pseudomonas aeruginosa consortium.

The International Pseudomonas aeruginosa Consortium is sequencing over 1000 genomes and building an analysis pipeline for the study of Pseudomonas genome evolution, antibiotic resistance and virulence genes. Metadata, including genomic and phenotypic data for each isolate of the collection, are available through the International Pseudomonas Consortium Database (http://ipcd.ibis.ulaval.ca/). Here, we present our strategy and the results that emerged from the analysis of the first 389 genomes. With as yet unmatched resolution, our results confirm that P. aeruginosa strains can be divided into three major groups that are further divided into subgroups, some not previously reported in the literature. We also provide the first snapshot of P. aeruginosa strain diversity with respect to antibiotic resistance. Our approach will allow us to draw potential links between environmental strains and those implicated in human and animal infections, understand how patients become infected and how the infection evolves over time as well as identify prognostic markers for better evidence-based decisions on patient care.

Characterization of the diffusion of polyethylene glycol in Streptococcus mutans biofilms by Raman microspectroscopy.

We used Raman microspectroscopy to investigate in situ the spatial distribution of the biomass in Streptococcus mutans biofilms. We used the CH stretching band to probe the organic matter and the area of the OH stretching band as an internal intensity standard, the biofilms being highly hydrated. The size of the biofilm regions that were mapped was 300 x 300 microm. We also recorded, in the confocal mode, the z profiles describing the biomass distribution as a function of depth in the biofilms. In our growth conditions, the biofilm is described as an approximately 75 microm thick mat completely covering the surface and includes columnar clusters with a diameter of approximately 100 microm surrounded by voids filled with water. Raman mapping was also used to examine the diffusion of HOD and polyethylene glycol with a molar mass of 10,000 (PEG-10k) in the biofilms. This study establishes that HOD can diffuse practically everywhere in the biofilms but that the penetration of PEG-10k is limited. There is a correlation between the restricted penetration of the macromolecule and the biomass content in the different regions of the biofilms. The method presented here provides a convenient approach to determine the diffusion of molecules, including antibacterials, in bacterial biofilms.

Reassessing the presence of Candida albicans in denture-related stomatitis.

OBJECTIVE: The aim of this study was to reevaluate the link between Candida albicans and denture-related stomatitis according to a modified Newton classification, which reflects the classic types of inflammation as well as the extent to which the tissue is affected. STUDY DESIGN: Two groups of denture wearers were evaluated for denture-related stomatitis. The presence and number of yeasts on the dentures, the identification of the Candida species present, and the amount of plaque coverage were determined. Putative risk factors were included in the study. Relations between these variables and stomatitis were analyzed statistically. RESULTS: According to Newton's classification, the presence of yeast on the denture was not linked to whether subjects had stomatitis. But with our classification, higher prevalence of yeast carriers, yeast colony number, and plaque coverage were found on the dentures of individuals with the most extensive inflammation, regardless of Newton type. Among risk factors evaluated, wearing dentures at night and smoking were associated with the most extensive inflammation. We also demonstrated that the presence of C albicans as well as the cohabitation of different Candida species was more frequent in denture-related stomatitis. The differences were statistically significant. CONCLUSIONS: Statistical analysis of microbiologic data from different denture-related stomatitis categories according to our modified classification showed that the presence of yeast on dentures was significantly associated with the extent of the inflammation, rather than with the Newton type. Our findings suggest that the inflammatory process of stomatitis favors the colonization of Candida. These results could have new implications for diagnosis and management of the condition.

A review of the science regarding dental unit waterlines.

BACKGROUND: The National Institute of Dental and Craniofacial Research, or NIDCR; the American Dental Association, or ADA; and the Organization for Safety & Asepsis Procedures, or OSAP, sponsored a workshop on the topic of dental unit waterlines, or DUWLs, on Sept. 29, 2000, at the National Institutes of Health in Bethesda, Md. These organizations invited a group of experts from the ADA, NIDCR, OSAP, the U.S. Food and Drug Administration, the Centers for Disease Control and Prevention, the U.S. Department of Defense, academia and private industry to participate. TYPES OF STUDIES REVIEWED: The sponsors asked the participants to critically review the scientific literature on the subject in an attempt to determine the evidence basis for management of DUWL contamination and potential health risks, if any, in dental procedures. The ultimate goal of the workshop was to determine if a research agenda in the area of DUWLs should be pursued and what questions such an agenda should involve. RESULTS: The workshop yielded four questions that need to be addressed in future research: What is the safest and most effective agent(s)/device(s) for achieving microbial levels of no more than 200 colony-forming units per milliliter, or CFU/mL, in the effluent dental water? How should these products be evaluated and by whom? What are the adverse health effects, if any, of chronic exposure to dental bioaerosol or to the agents introduced into the dental unit to treat the waterlines for both dental staff members and patients? How could these health issues be evaluated? CLINICAL IMPLICATIONS: Developing evidence-based parameters for the management of biofilm contamination that are efficacious and cost-effective will allow clinicians to meet in proposed ADA standard of no more than 200 CFU/mL of effluent water.