Combinatorial actions of IL-22 and IL-17 drive optimal immunity to oral candidiasis through SPRRs.

Oropharyngeal candidiasis (OPC) is the most common human fungal infection, arising typically from T cell immune impairments. IL-17 and IL-22 contribute individually to OPC responses, but here we demonstrate that the combined actions of both cytokines are essential for resistance to OPC. Mice lacking IL-17RA and IL-22RA1 exhibited high fungal loads in esophagus- and intestinal tract, severe weight loss, and symptoms of colitis. Ultimately, mice succumbed to infection. Dual loss of IL-17RA and IL-22RA impaired expression of small proline rich proteins (SPRRs), a class of antimicrobial effectors not previously linked to fungal immunity. Sprr2a1 exhibited direct candidacidal activity in vitro, and Sprr1-3a-/- mice were susceptible to OPC. Thus, cooperative actions of Type 17 cytokines mediate oral mucosal anti-Candida defenses and reveal a role for SPRRs.

Efficacy of a novel three-step decontamination protocol for titanium-based dental implants: An in vitro and in vivo study.

AIM: The aim of the study was to evaluate several mechanical and chemical decontamination methods associated with a newly introduced biofilm matrix disruption strategy for biofilm cleaning and preservation of implant surface features. MATERIALS AND METHODS: Titanium (Ti) discs were obtained by additive manufacturing. Polymicrobial biofilm-covered Ti disc surfaces were decontaminated with mechanical [Ti curette, Teflon curette, Ti brush, water-air jet device, and Er:YAG laser] or chemical [iodopovidone (PVPI) 0.2% to disrupt the extracellular matrix, along with amoxicillin; minocycline; tetracycline; H2 O2 3%; chlorhexidine 0.2%; NaOCl 0.95%; hydrocarbon-oxo-borate-based antiseptic] protocols. The optimal in vitro mechanical/chemical protocol was then tested in combination using an in vivo biofilm model with intra-oral devices. RESULTS: Er:YAG laser treatment displayed optimum surface cleaning by biofilm removal with minimal deleterious damage to the surface, smaller Ti release, good corrosion stability, and improved fibroblast readhesion. NaOCl 0.95% was the most promising agent to reduce in vitro and in vivo biofilms and was even more effective when associated with PVPI 0.2% as a pre-treatment to disrupt the biofilm matrix. The combination of Er:YAG laser followed by PVPI 0.2% plus NaOCl 0.95% promoted efficient decontamination of rough Ti surfaces by disrupting the biofilm matrix and killing remnants of in vivo biofilms formed in the mouth (the only protocol to lead to ~99% biofilm eradication). CONCLUSION: Er:YAG laser + PVPI 0.2% + NaOCl 0.95% can be a reliable decontamination protocol for Ti surfaces, eliminating microbial biofilms without damaging the implant surface.

Peri-implantitis: Knowledge and attitudes of implantology clinicians regarding the disease management: Peri-implantitis knowledge.

OBJECTIVES: Since peri-implantitis is an increasing and prevalent concern in clinical practice and there is no consensus regarding the best therapeutic protocol, this study evaluated the knowledge and behaviours of dentists working in Implantology regarding implant-related infections modulating factors and therapeutic protocols used in the management of peri-implantitis. METHODS: Cross-sectional study was conducted with 86 Brazilian Implantology clinicians. Data were collected using a structured and online questionnaire evaluating socioeconomic characteristics, education, work/clinical practice, knowledge and attitudes regarding the risk factors and management of peri-implantitis. The reliability of the questionnaire was evaluated by test-retest technique. The questionnaire was developed based on the last consensus on peri-implant diseases (2018) and the current evidence related to implant-related infections. Descriptive, bivariate and logistic regression analyses were conducted adopting a significance level of 5%. RESULTS: In this study, 89.5% of included dentists reported that already treated patients with peri-implantitis. Approximately 80% of dentists use antibiotics and mouth rinses during the treatment, and surgical procedures seem the main choice to treat peri-implantitis (91.8%) by dentists. As a preventive approach, 94.2% of dentists reported that routinely assessed biofilm accumulation in the follow-up visits after implant placement. Logistic regression showed that the self-reported ability to treat peri-implantitis was statistically (p < 0.05) higher among dentists who reported abilities to diagnose the disease and use laser for peri-implantitis treatment. CONCLUSION: Dentists working in Implantology have a good level of knowledge and behaviors in the management of peri-implantitis. However, the lack of consensus regarding the best treatment protocols may reflect dentist's behaviours because different treatment protocols have been used by evaluated clinicians.

Effect of Processing Methods of Human Saliva on the Proteomic Profile and Protein-Mediated Biological Processes.

The use of saliva as a protein source prior to microbiological and biological assays requires previous processing. However, the effect of these processing methods on the proteomic profile of saliva has not been tested. Stimulated human saliva was collected from eight healthy volunteers. Non-processed saliva was compared with 0.22 µm filtered, 0.45 µm filtered, and pasteurized saliva, by liquid chromatography-mass spectrometry. Data are available via ProteomeXchange with identifier PXD039248. The effect of processed saliva on microbial adhesion was tested using bacterial and fungus species and in biological cell behavior using HaCaT immortalized human keratinocytes. Two hundred and seventy-eight proteins were identified in non-processed saliva, of which 54 proteins (≈19%) were exclusive. Saliva processing reduced identified proteins to 222 (≈80%) for the 0.22 µm group, 219 (≈79%) for the 0.45 µm group, and 201 (≈72%) for the pasteurized saliva, compared to non-processed saliva. The proteomic profile showed similar molecular functions and biological processes. The different saliva processing methods did not alter microbial adhesion (ANOVA, p > 0.05). Interestingly, pasteurized saliva reduced keratinocyte cell viability. Saliva processing methods tested reduced the proteomic profile diversity of saliva but maintained similar molecular functions and biological processes, not interfering with microbial adhesion and cell viability, except for pasteurization, which reduced cell viability.

Polymicrobial biofilms related to dental implant diseases: unravelling the critical role of extracellular biofilm matrix.

Biofilms are complex tri-dimensional structures that encase microbial cells in an extracellular matrix comprising self-produced polymeric substances. The matrix rich in extracellular polymeric substance (EPS) contributes to the unique features of biofilm lifestyle and structure, enhancing microbial accretion, biofilm virulence, and antimicrobial resistance. The role of the EPS matrix of biofilms growing on biotic surfaces, especially dental surfaces, is largely unravelled. To date, there is a lack of a broad overview of existing literature concerning the relationship between the EPS matrix and the dental implant environment and its role in implant-related infections. Here, we discuss recent advances in the critical role of the EPS matrix on biofilm growth and virulence on the dental implant surface and its effect on the etiopathogenesis and progression of implant-related infections. Similar to other biofilms associated with human diseases/conditions, EPS-enriched biofilms on implant surfaces promote microbial accumulation, microbiological shift, cross-kingdom interaction, antimicrobial resistance, biofilm virulence, and, consequently, peri-implant tissue damage. But intriguingly, the protagonism of EPS role on implant-related infections and the development of matrix-target therapeutic strategies has been neglected. Finally, we highlight the need for more in-depth analyses of polymicrobial interactions within EPS matrix and EPS-targeting technologies' rationale for disrupting the complex biofilm microenvironment with more outstanding translation to implant applications in the near future.

In-vitro polymicrobial oral biofilm model represents clinical microbial profile and disease progression during implant-related infections.

AIM: Clinically relevant in-vitro biofilm models are essential and valuable tools for mechanistically dissecting the etiopathogenesis of infectious diseases and test new antimicrobial therapies. Thus, the aim of this study was to develop and test a clinically relevant in-vitro oral polymicrobial biofilm model that mimics implant-related infections in terms of microbial profile. METHODS AND RESULTS: For this purpose, 24-well plate system was used to model oral biofilms, using three different microbial inoculums to grow in-vitro biofilms: (1) human saliva from periodontally healthy patients; (2) saliva as in inoculum 1 + Porphyromonas gingivalis strain; and (3) supra and subgingival biofilm collected from peri-implant sites of patients diagnosed with peri-implantitis. Biofilms were grown to represent the dynamic transition from an aerobic to anaerobic community profile. Subsequently, biofilms were collected after each phase and evaluated for microbiological composition, microbial counts, biofilm biomass, structure, and susceptibility to chlorhexidine (CHX). Results showed higher live cell count (P < .05) for biofilms developed from patients' biofilm inoculum, but biomass volume, dry weight, and microbiological composition were similar among groups (P > .05). Interestingly, according to the checkerboard DNA-DNA hybridization results, the biofilm developed from stimulated human saliva exhibited a microbial composition more similar to the clinical subgingival biofilm of patients with peri-implantitis, with proportions of the main pathogens closer to those found in the disease. In addition, biofilm developed using saliva as inoculum was shown to be susceptible to CHX with significant reduction in bacteria compared with biofilms without exposure to CHX (P < .05). CONCLUSION: The findings suggested that the in-vitro polymicrobial biofilm developed from human saliva as inoculum is a suitable model and clinically relevant tool for mimicking the microbial composition of implant-related infections.

Clinical efficacy of triclosan-containing toothpaste in peri-implant health: A systematic review and meta-analysis of randomized clinical trials.

STATEMENT OF PROBLEM: Recent evidence suggests that toothpaste containing 0.3% triclosan (TCS) is more effective than regular toothpaste in improving clinical periodontal conditions. However, a consensus on whether TCS favors a healthy peri-implant environment is limited. PURPOSE: The purpose of this systematic review and meta-analysis of randomized clinical trials was to determine the effects of TCS-containing toothpaste on dental implant health based on clinical, immunological, and microbiological parameters, as well as on reported adverse events. MATERIAL AND METHODS: Clinical studies comparing peri-implant conditions in participants by using TCS toothpaste versus conventional fluoride toothpaste (control) were extracted from 9 databases. The studies were assessed with the Cochrane risk-of-bias tool for randomized clinical trials (RoB 2). Datasets for bleeding on probing (BOP), probing depth (PD), clinical attachment level (CAL), gingival index (GI), plaque index (PI), osteo-immunoinflammatory mediators, and bacterial load were plotted, and the standard mean difference (SMD) quantitative analysis was applied by using the Rev Man 5.3 software program. Adverse effects reported by the studies were also tabulated. The certainty of evidence was assessed by using the grading of recommendations assessment, development, and evaluation approach. RESULTS: Six studies were included in the meta-analyses. BOP was higher in the control group than in the TCS toothpaste group at 3 months (SMD -0.59 [-1.11, -.07] P=.002, I2=77%) and 6 months (SMD -0.59 [-0.83, -0.34] P=.009, I2=79%). PD (SMD -0.04 [-0.08, -0.00] P=.04, I2=0%) was also deeper in the control group versus TCS toothpaste at 6 months (SMD -0.41 [-0.73, -0.10] P=.04, I2=77%). CAL, GI, and PI did not differ between groups (P>.05). Among the osteo-immunoinflammatory mediators, IL-10 levels increased, and IL-1ß and osteoprotegerin levels decreased in the TCS toothpaste group (P<.05). Microbiological findings found that TCS toothpaste prevented the growth of periodontal pathogens, specifically in up to approximately 20% of the Prevotella intermedia. Adverse effects were not reported after toothbrushing in either group. However, most studies had "some" or "high" risk of bias, and the certainty of the evidence was considered to be "very low." CONCLUSIONS: Most studies were short-term (3 and 6 months) analyses, and the results found that, although TCS-containing toothpaste had positive osteo-immunoinflammatory and microbiologic results, clinical parameters, including CAL, GI, and PI, were not influenced.

Oral microbial colonization on titanium and polyetheretherketone dental implant healing abutments: An in vitro and in vivo study.

STATEMENT OF PROBLEM: Although polyetheretherketone (PEEK) implant healing abutments have become popular because of their esthetic, mechanical, and chemical properties, studies analyzing oral polymicrobial adhesion to PEEK abutments are lacking. PURPOSE: The purpose of this in vitro and in vivo study was to evaluate oral microbial adhesion and colonization on titanium (Ti) and PEEK healing abutments. MATERIAL AND METHODS: Ti (N=35) and PEEK substrates (N=35) were evaluated in vitro in terms of the initial adhesion (1 hour) or biofilm accumulation (48 hours) of Candida albicans and a polymicrobial inoculum using stimulated human saliva to mimic a diverse oral microbiome. Surface decontamination ability was evaluated after 24 hours of in vitro biofilm formation after exposure to an erbium-doped yttrium aluminum garnet (Er:YAG) laser. Conventional and flowable composite resin veneering on PEEK was also tested for microbial adhesion. In addition, an in vivo model with 3 healthy volunteers was conducted by using a palatal appliance containing the tested materials (3 or 4 specimens of each material per appliance) for 2 days to evaluate the effect of substrate on the microbial profile. Biofilms were evaluated by live cell counts and scanning electron microscopy images, and the microbial profile by Checkerboard deoxyribonucleic acid (DNA)-DNA hybridization. The t test and Mann-Whitney test were used to compare the groups (α=.05). RESULTS: PEEK and Ti materials showed similar fungal adhesion (P>.05). Although the PEEK surface limited the initial in vitro polymicrobial adhesion (approximately 2 times less) compared with Ti (P=.040), after 48 hours of biofilm accumulation, the microbial load was statistically similar (P=.209). Er:YAG laser decontamination was more effective on PEEK than on Ti surfaces, reducing approximately 11 times more microbial accumulation (P=.019). Both composite resins tested showed similar microbial adhesion (1 hour). In vivo, the PEEK material showed reduced levels of 6 bacterial species (P<.05), including the putative pathogen Treponema denticola. CONCLUSIONS: Although PEEK and Ti had similar bacterial and fungus biofilm attachment and accumulation, PEEK promoted a host-compatible microbial profile with a significantly reduced T. denticola load.

Knowledge of bidirectional relationship between diabetes and periodontal disease among diabetes patients: A systematic review.

INTRODUCTION: The bidirectional relationship between diabetes mellitus and periodontal disease has been reported in the literature, suggesting that poor glycemic control is strongly associated with increased risk of developing periodontal disease. Therefore, this systematic review evaluated the level of knowledge of this bidirectional relationship among patients with diabetes. METHODS: This systematic review (protocol CRD42018117902) was conducted according to PRISMA guidelines. The following databases were considered: Medline/PubMed, Scopus, and Web of Science. Search strategy (April 05th , 2021) considered proper combination of keywords and eligibility criteria. The quality of studies was evaluated using the Appraisal tool for Cross-Sectional Studies (AXIS). RESULTS: Among the 328 records identified in the initial search, 24 studies were selected, considering a total of 8,693 patients. All studies used a cross-sectional design. Among the included studies, only five showed prevalence of knowledge higher than 50%, ranging from 5.8% to 75.9%. Interestingly, 58.0% of patients reported that they brush their teeth at least 1x/day, but only four studies reported that the dentist was the main source of information. In terms of methodology and result quality, just one study clearly showed all information evaluated by the AXIS tool. Most of studies did not report sample size calculations and did not used validated questionnaires to assess patient knowledge. CONCLUSION: The results show that less than half of people with diabetes have knowledge about their increased risk for periodontal disease, and often the dentist is not the main source of information to motivate them.

Do the progression of experimentally induced gingivitis and peri-implant mucositis present common features? A systematic review of clinical human studies.

This systematic review evaluated the features of the progression of experimentally induced gingivitis and peri-implant mucositis in humans. Included were studies that evaluated clinical, immunological, or microbiological responses between experimentally induced gingivitis and peri-implant mucositis in periodontally healthy patients. A total of 887 articles were initially identified, but only 12 were included in the final analysis. Implants accumulate less biofilm and suffer the most heterogeneous alterations in the microbiota, in the abstinence of oral hygiene, compared with the tooth. Interestingly, although dental implants presented less biofilm accumulation, the peri-implant mucosa showed a more exacerbated clinical response than the gingival tissue. The risk of bias of the selected studies was moderate to low, with one study presenting serious risk. The progression events of peri-implant mucositis were similar to those of experimental gingivitis but led to a different host response. This review was registered in the PROSPERO database CRD420201 123360.

Filling Materials Efficacy on Preventing Biofilm Formation Inside Screw Access Channels of Implant Abutments.

The choice of the material used to fill screw access channels in implant-supported prostheses depends, in most cases, on operator's preference, without considering the susceptibility of biofilm colonization. Therefore, the aim of this study was to determine and compare the total amount of biofilm formed on different materials used to fill screw access channels in implant abutments. For this propose, titanium implant analogs were attached on abutments and divided into 5 groups: positive control (no filling material); negative control (closed with resin); and filled with cotton, gutta-percha, or polytetrafluoroethylene (PTFE). The analogs with attached abutments were then immersed in a brain heart infusion medium containing Candida albicans (strain 10231 from American Type Culture Collection [ATCC]) and incubated aerobically at 37°C with gentle agitation. After 15 days, materials were removed, and total viable biofilm on each material was quantified by methyl tetrazolium reduction assay at 490 nm. All experiments were performed in triplicate. Data were processed by IBM SPSS Statistic software using 1-way analysis of variance and Bonferroni post hoc tests to analyze differences between groups, with an overall significance level of P < .001. A significant difference was observed between cotton and gutta-percha (P < .017) and between cotton and PTFE (P < .025). However, there was no statistical difference between gutta-percha and PTFE (P > .050). Thus, this in vitro experiment showed that gutta-percha and PTFE presented lower biofilm formation compared with cotton when used to fill screw access channels. These results can provide a basis for future clinical studies that can be a guide to decreasing the occurrence of gaps and bacterial growth inside the implant/abutment attachment site. In addition, controlled in vivo studies are necessary to confirm the clinical viability of findings of this study.

Candida albicans induces mucosal bacterial dysbiosis that promotes invasive infection.

Infectious complications are a common cause of morbidity and mortality in cancer patients undergoing chemotherapy due to increased risk of oral and gastrointestinal candidiasis, candidemia and septicemia. Interactions between C. albicans and endogenous mucosal bacteria are important in understanding the mechanisms of invasive infection. We published a mouse intravenous chemotherapy model that recapitulates oral and intestinal mucositis, and myelosuppression in patients receiving 5-fluorouracil. We used this model to study the influence of C. albicans on the mucosal bacterial microbiome and compared global community changes in the oral and intestinal mucosa of the same mice. We validated 16S rRNA gene sequencing data by qPCR, in situ hybridization and culture approaches. Mice receiving both 5Fu and C. albicans had an endogenous bacterial overgrowth on the oral but not the small intestinal mucosa. C. albicans infection was associated with loss of mucosal bacterial diversity in both sites with indigenous Stenotrophomonas, Alphaproteobacteria and Enterococcus species dominating the small intestinal, and Enterococcus species dominating the oral mucosa. Both immunosuppression and Candida infection contributed to changes in the oral microbiota. Enterococci isolated from mice with oropharyngeal candidiasis were implicated in degrading the epithelial junction protein E-cadherin and increasing the permeability of the oral epithelial barrier in vitro. Importantly, depletion of these organisms with antibiotics in vivo attenuated oral mucosal E-cadherin degradation and C. albicans invasion without affecting fungal burdens, indicating that bacterial community changes represent overt dysbiosis. Our studies demonstrate a complex interaction between C. albicans, the resident mucosal bacterial microbiota and the host environment in pathogenesis. We shed significant new light on the role of C. albicans in shaping resident bacterial communities and driving mucosal dysbiosis.

Optimizing citric acid protocol to control implant-related infections: An in vitro and in situ study.

OBJECTIVE: The present study aimed to establish an optimized protocol for biofilm removal from titanium (Ti) surfaces using citric acid (CA) solutions. BACKGROUND: Biofilm accumulation is the main factor to trigger peri-implant infections and to increase the risk of treatment failures. Although CA has been suggested as the anti-infective agent with highest potential for biofilm removal on Ti, there is no consensus that CA could improve the anti-infective treatment and its effect. METHODS: Physical and chemical alterations, electrochemical behavior, cytotoxicity, and antimicrobial effect of CA on Ti discs were evaluated using four concentrations (1, 10, 20, and 40%) and two application methods (immersion and rubbing). Negative control using 0.9% NaCl was used in all experiments. To evaluate whether different application times can have similar response, polymicrobial biofilm (microcosm model) was formed on Ti and treated with CA for 1, 2, 4, and 8 min. An in situ study was conducted to verify whether the established protocol is equally effective in biofilms formed on machined and sandblasted, large-grit, and acid-etched (SLA) Ti surfaces. RESULTS: CA 40% induced significantly higher surface alterations observed by confocal images and profilometry. In general, rubbing protocol decreased the surface roughness and increased the wettability (p < 0.05), exhibiting better surface cleaning by biofilm removal. CA 10% presented no indirect cytotoxicity and, when applied by rubbing for 8 min, presented proper in vitro antibacterial action and potential corrosion inhibition. When CA 10% was rubbed on Ti surfaces for 4 min, it displayed optimum cleaning ability as 8 min, working equally to remove in situ biofilm on machined and SLA surfaces. CONCLUSIONS: The application of CA 10% by rubbing for at least 4 min demonstrated to be a promising protocol to eliminate biofilms formed in smooth and rougher surfaces, which could improve implant-related infection therapies.

Biofilm Interactions of Candida albicans and Mitis Group Streptococci in a Titanium-Mucosal Interface Model.

Streptococci from the mitis group (represented mainly by Streptococcus mitis, Streptococcus oralis, Streptococcus sanguinis, and Streptococcus gordonii) form robust biofilms with Candida albicans in different experimental models. These microorganisms have been found in polymicrobial biofilms forming on titanium biomaterial surfaces in humans with peri-implant disease. The purpose of this work was to study mutualistic interactions in biofilms forming on titanium and their effect on the adjacent mucosa, using a relevant infection model. Single and mixed biofilms of C. albicans and each Streptococcus species were grown on titanium disks. Bacterial and fungal biovolume and biomass were quantified in these biofilms. Organotypic mucosal constructs were exposed to preformed titanium surface biofilms to test their effect on secretion of proinflammatory cytokines and cell damage. C. albicans promoted bacterial biofilms of all mitis Streptococcus species on titanium surfaces. This relationship was mutualistic since all bacterial species upregulated the efg1 hypha-associated gene in C. albicans Mixed biofilms caused increased tissue damage but did not increase proinflammatory cytokine responses compared to biofilms comprising Candida alone. Interestingly, spent culture medium from tissues exposed to titanium biofilms suppressed Candida growth on titanium surfaces.IMPORTANCE Our findings provide new insights into the cross-kingdom interaction between C. albicans and Streptococcus species representative of the mitis group. These microorganisms colonize titanium-based dental implant materials, but little is known about their ability to cause inflammation and damage of the adjacent mucosal tissues. Using an in vitro biomaterial-mucosal interface infection model, we showed that mixed biofilms of each species with C. albicans enhance tissue damage. One possible mechanism for this effect is the increased fungal hypha-associated virulence gene expression we observed in mixed biofilms with these species. Interestingly, we also found that the interaction of multispecies biofilms with organotypic mucosal surfaces led to the release of growth-suppressing mediators of Candida, which may represent a homeostatic defense mechanism of the oral mucosa against fungal overgrowth. Thus, our findings provide novel insights into biofilms on biomaterials that may play an important role in the pathogenesis of mucosal infections around titanium implants.

Titanium particles and ions favor dysbiosis in oral biofilms.

OBJECTIVE: To evaluate the effect of titanium (Ti) particles and ions on oral biofilm growth and composition. BACKGROUND: Particles and ions of Ti released from dental implants can trigger unfavorable biological responses in human cells. However, their effect on oral biofilms composition has not been tested. METHODS: In this blind in situ study, volunteers wore a palatal appliance containing Ti disks for 7 days to allow biofilm formation. Disks were then collected and biofilms were treated, in vitro, with Ti particles (0.75% and 1%), ions (10 and 20 ppm), or a combination of both (1% particles + 20 ppm ions). Biofilms exposed only to medium was used as control group. After 24 hours, biofilms were collected and analyzed by checkerboard DNA-DNA hybridization. Direct effects of Ti particles and ions on biofilm/cellular morphology were evaluated by transmission electron microscopy (TEM). RESULTS: Ti particles affected biofilm composition, increasing population of four bacterial species (P < .05), while Ti ions showed higher levels of putative pathogens from the orange complex with reduction in species from the yellow complex (P < .05), compared with control. The combination of particles + ions increased green complex and reduced yellow complex proportions (P < .05). TEM showed clusters of particles agglomerated in extracellular environment, while Ti ions were precipitated in both extracellular and intracellular sites. CONCLUSIONS: Ti products, especially Ti ions, have the potential to change the microbiological composition of biofilms formed on Ti surfaces. Therefore, the presence of Ti products around dental implants may contribute to microbial dysbiosis and peri-implantitis.

Extracellular biofilm matrix leads to microbial dysbiosis and reduces biofilm susceptibility to antimicrobials on titanium biomaterial: An in vitro and in situ study.

OBJECTIVES: To test the role of exopolysaccharide (EPS) polymers matrix to modulate the composition/virulence of biofilms growing on titanium (Ti) surfaces, the effect on antibiotic susceptibility, and whether a dual-targeting therapy approach for disrupted EPS matrix could improve the antimicrobial effect. MATERIALS AND METHODS: A microcosm biofilm model using human saliva as inoculum was used, and the microbial composition was assessed by checkerboard DNA-DNA hybridization. EPS-enriched biofilms virulence was tested using fibroblast monolayer. Povidone-iodine (PI) was used as EPS-targeting agent followed by amoxicillin + metronidazole antibiotic to reduce bacterial biomass using an in situ model. RESULTS: An EPS-enriched environment, obtained by sucrose exposure, promoted bacterial accumulation and led to a dysbiosis on biofilms, favoring the growth of Streptococcus, Fusobacterium, and Campylobacter species and even strict anaerobic species related to peri-implant infections, such as Porphyromonas gingivalis and Tannerella forsythia (~3-fold increase). EPS-enriched biofilm transitioned from a commensal aerobic to a pathogenic anaerobic profile. EPS increased biofilm virulence promoting higher host cell damage and reduced antimicrobial susceptibility, but the use of a dual-targeting approach with PI pre-treatment disrupted EPS matrix scaffold, increasing antibiotic effect on in situ biofilms. CONCLUSION: Altogether, our data provide new insights of how EPS matrix creates an environment that favors putative pathogens growth and shed light to a promising approach that uses matrix disruption as initial step to potentially improve implant-related infections treatment.

Proteomic profile of the saliva and plasma protein layer adsorbed on Ti-Zr alloy: the effect of sandblasted and acid-etched surface treatment.

Titanium-zirconium (Ti-Zr) alloy has been widely used as a biomaterial for implant devices, and it is commonly treated by sandblasting followed by acid etching (SLA) to improve biological responses. Although protein adsorption is the first biological response, the effect of this SLA treatment on the proteomic profile of proteins adsorbed from saliva and blood plasma has not been tested. In this study, the proteomic profile was evaluated by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). Streptococcus sanguinis was used to test whether the protein layer affects bacterial adhesion. SLA treatment affected the proteomic profile, showing exclusive proteins adsorbed from saliva (14) and plasma (3). However, both groups exhibited close patterns of intensity for common proteins, molecular functions and biological processes mediated by proteins. Interestingly, Ti-ZrSLA showed higher bacterial adhesion (∼1.9 fold over) for the surface coated with plasma proteins. Therefore, SLA treatment of Ti-Zr alloy changed the proteomic profile, which may affect bacterial adhesion.

In vitro evaluation of physicochemical properties of soft lining resins after incorporation of chlorhexidine.

STATEMENT OF PROBLEM: Incorporating chlorhexidine into soft lining materials has been suggested to reduce biofilm development on the material surface and treat denture stomatitis. However, evaluation of the physicochemical properties of this material is necessary. PURPOSE: The purpose of this in vitro study was to evaluate the physicochemical properties of resin-based denture soft lining materials modified with chlorhexidine diacetate (CDA). MATERIAL AND METHODS: Two soft lining resins were tested, one based on polymethyl methacrylate (PMMA) and the other on polyethyl methacrylate (PEMA), into which 0.5%, 1.0%, or 2.0% of CDA was incorporated; the control group had no CDA. The specimens were stored for 2 hours, 48 hours, 7, 14, 21, and 28 days and then analyzed for polymer crystallinity, Shore A hardness, degree of monomer conversion, residual monomer leaching, and CDA release. Data were analyzed by using a 3-way ANOVA and the Tukey HSD test (α=.05). RESULTS: The polymer crystallinity of PEMA and PMMA did not change after CDA incorporation. Shore A hardness increased over time, but not for any CDA concentrations tested after 28 days (P>.05). Considering the degree of conversion, PMMA-based resin showed no statistically significant difference (P>.05). However, PEMA-based resin showed a significant decrease (P<.05), which was reflected in a significant increase in residual monomer leaching from PEMA-based resin with the incorporation of 0.5% and 1.0% CDA (P<.05), mainly in the first 48 hours. PMMA-based resin showed no change in monomer leaching (P>.05). For both resins, the CDA release kinetics were related to monomer leaching; for PEMA-based resin, the values were significantly higher in the first 48 hours (P<.05), and for PMMA-based resin, the values were more sustained up to the last day of analysis. CONCLUSIONS: The incorporation of CDA did not affect the physicochemical properties of soft resins. The properties of PMMA were better than those of PEMA.

The Relationship of Candida albicans with the Oral Bacterial Microbiome in Health and Disease.

Candida albicans is an opportunistic pathogen colonizing the oropharyngeal, esophageal, and gastrointestinal mucosa in most healthy humans. In immunocompromised hosts, this fungal organism can cause mucosal candidiasis in these sites. C. albicans also causes fungemia, a serious consequence of cancer cytotoxic chemotherapy, which is thought to develop from fungal translocation through compromised mucosal barriers. Changes in endogenous bacterial population size or composition as well as changes in the host environment can transform fungal commensals into opportunistic pathogens in the upper and lower GI tract. Pioneering studies from our group have shown that a ubiquitous oral commensal of the mitis streptococcal group (Streptococcus oralis) has a mutualistic relationship with C. albicans, with C. albicans enabling streptococcal biofilm growth at mucosal sites, and S. oralis facilitating invasion of the oral and esophageal mucosa by C. albicans. In these studies, we used a cortisone-induced immunosuppression mouse model. More recently, the development of a novel mouse chemotherapy model has allowed us to examine the interactions of C. albicans with the endogenous bacterial microbiota in the oral and small intestinal mucosa, two sites adversely affected by cytotoxic chemotherapy. In this model, oral inoculation with C. albicans causes severe dysbiosis in the mucosal bacterial composition in both sites. We also found that antibiotic treatment ameliorates invasion of the oral mucosa but aggravates dissemination through the intestinal mucosa. In this chapter, we discuss work from our laboratory and others examining the relationships of C. albicans with oral bacteria and their role in mucosal homeostasis or disease.

Streptococcus oralis and Candida albicans Synergistically Activate µ-Calpain to Degrade E-cadherin From Oral Epithelial Junctions.

Streptococcus oralis forms robust mucosal biofilms with Candida albicans that have increased pathogenic potential. In this study, using oral epithelial cultures, organotypic oral mucosal constructs, and a mouse model of oral infection, we demonstrated that S. oralis augmented C. albicans invasion through epithelial junctions. C. albicans and S. oralis decreased epithelial E-cadherin levels by synergistically increasing µ-calpain, a proteolytic enzyme that targets E-cadherin. In the mouse coinfection model this was accompanied by increased fungal kidney dissemination. Coinfection with a secreted aspartyl protease (sap) mutant sap2456 and S. oralis increased µ-calpain and triggered mucosal invasion and systemic dissemination, suggesting that fungal protease activity is not required for invasion during coinfection. We conclude that C. albicans and S. oralis synergize to activate host enzymes that cleave epithelial junction proteins and increase fungal invasion.