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.

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.

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.

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.

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.

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.

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.

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.

Phase 1 clinical trial on the effect of palatal brushing on denture stomatitis.

PURPOSE: This study aimed to assess the efficacy of palatal brushing in the treatment of denture stomatitis. MATERIALS AND METHODS: After screening 143 individuals with a potential diagnosis of denture stomatitis, 48 patients (mean age: 66.0 ± 11.2 years) were enrolled in a two-center phase 1 clinical trial with a one-group pretest/posttest design. The intervention of interest was manual palatal brushing after each meal and before bedtime. Clinical and microbiologic examinations were performed at baseline and 1 and 3 months after treatment. Additional data were obtained using a validated questionnaire. The primary and secondary outcomes were the remission of denture stomatitis and diminution of Candida colony-forming units (CFUs), respectively. Descriptive and nonparametric statistical tests were conducted to analyze the data. RESULTS: At the 3-month follow-up, denture stomatitis was completely cured in 10.4% of the participants, and 70.8% of denture wearers showed improvement in the clinical signs of denture stomatitis. There was a significant reduction in the area and severity of the palatal inflammation (P < .0001). The effect size ranged from medium to large (0.34 to 0.54) depending on the classification used for the diagnosis of denture stomatitis. A significant reduction in the number of Candida CFUs isolated from the palatal mucosa and dentures (P ≤ .05) was observed. CONCLUSIONS: The results of this study suggest that palatal brushing is an effective treatment of denture stomatitis.

Oxidative nanopatterning of titanium generates mesoporous surfaces with antimicrobial properties.

Mesoporous surfaces generated by oxidative nanopatterning have the capacity to selectively regulate cell behavior, but their impact on microorganisms has not yet been explored. The main objective of this study was to test the effects of such surfaces on the adherence of two common bacteria and one yeast strain that are responsible for nosocomial infections in clinical settings and biomedical applications. In addition, because surface characteristics are known to affect bacterial adhesion, we further characterized the physicochemical properties of the mesoporous surfaces. Focused ion beam (FIB) was used to generate ultrathin sections for elemental analysis by energy-dispersive X-ray spectroscopy (EDS), nanobeam electron diffraction (NBED), and high-angle annular dark field (HAADF) scanning transmission electron microscopy (STEM) imaging. The adherence of Staphylococcus aureus, Escherichia coli and Candida albicans onto titanium disks with mesoporous and polished surfaces was compared. Disks with the two surfaces side-by-side were also used for direct visual comparison. Qualitative and quantitative results from this study indicate that bacterial adhesion is significantly hindered by the mesoporous surface. In addition, we provide evidence that it alters structural parameters of C. albicans that determine its invasiveness potential, suggesting that microorganisms can sense and respond to the mesoporous surface. Our findings demonstrate the efficiency of a simple chemical oxidative treatment in generating nanotextured surfaces with antimicrobial capacity with potential applications in the implant manufacturing industry and hospital setting.

Pseudomonas aeruginosa isolates from dental unit waterlines can be divided in two distinct groups, including one displaying phenotypes similar to isolates from cystic fibrosis patients.

Pseudomonas aeruginosa displays broad genetic diversity, giving it an astonishing capacity to adapt to a variety of environments and to infect a wide range of hosts. While many P. aeruginosa isolates of various origins have been analyzed, isolates from cystic fibrosis (CF) patients have received the most attention. Less is known about the genetic and phenotypic diversity of P. aeruginosa isolates that colonize other environments where flourishing biofilms can be found. In the present study, 29 P. aeruginosa isolates from dental unit waterlines and CF patients were collected and their genetic and phenotypes profiles were compared to determine whether environmental and clinical isolates are related. The isolates were first classified using the random amplified polymorphic DNA method. This made it possible to distribute the isolates into one clinical cluster and two environmental clusters. The isolates in the environmental cluster that were genetically closer to the clinical cluster also displayed phenotypes similar to the clinical isolates. The isolates from the second environmental cluster displayed opposite phenotypes, particularly an increased capacity to form biofilms. The isolates in this cluster were also the only ones harboring genes that encoded specific epimerases involved in the synthesis of lipopolysaccharides, which could explain their increased ability to form biofilms. In conclusion, the isolates from the dental unit waterlines could be distributed into two clusters, with some of the environmental isolates resembled the clinical isolates.

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.

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.

Molecular characterization of the polymicrobial flora in chronic rhinosinusitis.

INTRODUCTION: Conventional cultures have implicated Staphylococcus aureus (SA) and coagulase-negative Staphylococcus (CNS) as principal pathogens in chronic rhinosinusitis (CRS). These results are questioned by recent studies in which molecular probes implicate Haemophilus influenzae instead. OBJECTIVES: To identify all bacterial species present on sinonasal mucosa using molecular culture (bacterial tag-encoded FLX amplicon pyrosequencing [bTEFAP]) and to compare them with those identified with conventional methods. METHODS: A prospective study of 18 patients undergoing endoscopic sinus surgery for CRS and 9 control patients with pituitary adenomas was conducted. Per-operative mucosal biopsies were assessed with bTEFAP by sequencing the species-specific 16S ribosomal deoxyribonucleic acid (DNA) fragment for genetic identification of bacteria and then compared with simultaneous swab culture. RESULTS: Standard cultures showed mainly SA and CNS. Molecular cultures identified up to 20 organisms per sample. Surprisingly, anaerobic species predominated (Diaphorobacter and Peptoniphilus). SA was nevertheless detected in 50%. CONCLUSION: Molecular cultures such as bTEFAP are sensitive tools for bacterial identification in CRS and suggest that anaerobe involvement may be more frequent than presumed.

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.

Role of the ammonium group in the diffusion of quaternary ammonium compounds in Streptococcus mutans biofilms.

OBJECTIVES: Cetylpyridinium chloride (CPC), a quaternary ammonium compound, was shown to interact irreversibly with Streptococcus mutans biofilms, leading to a slow diffusion compared with poly(ethylene glycol) (PEG) molecules of similar size. The objective of this work is to determine if the retardation of CPC diffusion and its strong binding to biofilms is caused by interactions between the ammonium group of CPC and the exopolysaccharide (EPS) matrix. METHODS: First, we characterized the diffusion of two analogues of CPC in S. mutans biofilms: dodecylpyridinium chloride (DPC), carrying a shorter alkyl chain than CPC, and tetramethylene bispyridinium chloride (TMBPC), a compound carrying two positively charged ammonium groups. Second, we cultured biofilms with different densities of EPS and examined the impact of this density on the transport properties of CPC. The diffusion of these compounds was probed using infrared spectroscopy with attenuated total reflectance sampling. RESULTS: The diffusion of CPC, DPC and TMBPC in S. mutans biofilm is slower than that of PEG10k. In addition, TMBPC and DPC, as PEG10k, could be readily washed out from the biofilms while CPC association was practically irreversible. The penetration of CPC through the EPS matrix was found to be not significantly affected by the increased EPS density, whereas the penetration of PEG with a molar mass of 10k was considerably reduced. CONCLUSIONS: These results suggest that the interactions between the quaternary ammonium groups and the EPS matrices are not the prime contribution of the strong CPC binding, and the alkyl chain length plays a role in this association, likely through hydrophobic interactions.

The relationship of myceliated colonies of Candida albicans with denture stomatitis: an in vivo/in vitro study.

PURPOSE: Switching from smooth to myceliated colonies, a virulent trait of Candida albicans, may be implicated in Candida-associated denture stomatitis. The purpose of this study was to verify the relationship between the presence of denture stomatitis and the frequency of myceliated colonies of C. albicans isolates in denture wearers. Prevalence of denture stomatitis and influence of putative risk factors were also investigated. MATERIALS AND METHODS: Demographic and clinical data concerning oral and general health, smoking, denture status, diet, and hygiene habits of 40 complete maxillary denture wearers were collected from an autoevaluation questionnaire and oral examination. Detection of C. albicans in denture plaque and evaluation of hairy phenotype colonies were carried out on low nutrient media. Eleven subjects were followed-up at 1 month and 3 months after delivery of a new prosthesis. Results were statistically analyzed. RESULTS: Prevalence of denture stomatitis was 77.5%. No statistically significant relation was found between presence of stomatitis and frequency of myceliated colonies of C. albicans or presence of yeast. However, the study confirmed a statistically significant difference between Newton types IA and IIB stomatitis in relation to yeast colony-forming units, which were more than 300 times higher in type IIB. A direct relationship was observed between the presence of C. albicans and nocturnal denture use (P = .01) and an inverse relation was observed with brushing of the palate (P = .03). CONCLUSION: The ability of C. albicans strains isolated from dentures to produce myceliated colonies may not be directly involved in denture stomatitis.