Effect of hydroxyapatite nanoparticles coating of titanium surface on biofilm adhesion: An in vitro study.

AIM: To evaluate the adhesion of mono and duospecies biofilm on a commercially available dental implant surface coated with hydroxyapatite nanoparticles (nanoHA). MATERIAL AND METHODS: Titanium discs were divided into two groups: double acid-etched (AE) and AE coated with nanoHA (NanoHA). Surface characteristics evaluated were morphology, topography, and wettability. Mono and duospecies biofilms of Streptococcus sanguinis (S. sanguinis) and Candida albicans (C. albicans) were formed. Discs were exposed to fetal bovine serum (FBS) to form the pellicle. Biofilm was growth in RPMI1640 medium with 10% FBS and 10% BHI medium for 6 h. Microbial viability was evaluated using colony-forming unit and metabolic activity by a colorimetric assay of the tetrazolium salt XTT. Biofilm architecture and organization were evaluated by confocal laser scanning microscopy (CLSM) and scanning electron microscopy (SEM). RESULTS: AE surface had more pores, while NanoHA had even nanoHA crystals distribution. Roughness was similar (AE: 0.59 ± 0.07 µm, NanoHA: 0.69 ± 0.18 µm), but wettability was different (AE: Θw= 81.79 ± 8.55°, NanoHA: Θw= 53.26 ± 11.86°; P = 0.01). NanoHA had lower S. sanguinis viability in monospecies biofilm (P = 0.007). Metabolic activity was similar among all biofilms. In SEM both surfaces on C. albicans biofilm show a similar distribution of hyphae in mono and duospecies biofilms. AE surface has more S. sanguinis than the NanoHA surface in the duospecies biofilm. CLSM showed a large proportion of live cells in all groups. CONCLUSIONS: The nanoHA surface reduced the adhesion of S. sanguinis biofilm but did not alter the adhesion of C. albicans or the biofilm formed by both species.

Does Streptococcus oralis supernatant influence on the proliferation and virulence of Candida albicans?

OBJECTIVE: To evaluate the influence of Streptococcus oralis supernatant on the proliferation and virulence of Candida albicans. DESIGN: S. oralis supernatant was obtained by filtration of overnight cultures. Single or dual-species cultures of C. albicans and S. oralis were cultivated in both planktonic and biofilm-based models. Planktonic culture growth was measured, and mature biofilms formed on resin disks were collected to measure biofilm metabolic activity, total biomass, and cell counts. Hyphae formation (virulence factor) and biofilm thickness were analyzed by confocal laser scanning microscopy. Data were analyzed by a one-way ANOVA test followed by the Tukey posthoc test (α = 0.05). RESULTS: We found that S. oralis supernatant did not influence C. albicans proliferation in planktonic cultures. However, biofilms containing S. oralis supernatant showed higher cell metabolism than C. albicans monoculture biofilms and C. albicans-S. oralis dual-culture biofilms (p < 0.05). Though S. oralis supernatants did increase biofilm metabolic activity, they did not affect the total biomass and cell counts of C. albicans (p > 0.05). However, biofilm imaging revealed enhanced C. albicans hyphae formation in biofilms containing S. oralis supernatant compared to C. albicans monoculture biofilms. CONCLUSIONS: Secreted metabolites in S. oralis supernatant may contribute to C. albicans metabolism and virulence.

Epithelial responses and Candida albicans pathogenicity are enhanced in the presence of oral streptococci.

Experimental models that consider host-pathogen interactions are relevant for improving knowledge about oral candidiasis. The aim of this study was to assess the epithelial immune responses, Candida penetration of cell monolayers, and virulence during mixed species culture infections. Single species cultures of Candida albicans and mixed cultures (C. albicans, Streptococcus mutans, and Streptococcus sanguinis) were used to infect monolayers of HaCaT and FaDu ATCC HTB-43 cells for 12 h. After infection, IL-18 and IL-34 gene expression was measured to assess epithelial cell immune responses, and lactate dehydrogenase (LDH) activity was measured as an indicator of cell damage. Microscopy determined C. albicans morphology and penetration of fungal cells through the keratinocyte monolayer. Monolayers devoid of infection served as controls. Data were analyzed by an ANOVA one-way test followed by Tukey's post-hoc test (α = 0.05). The results found that IL-18 and IL-34 gene expression and LDH activity were significantly (p < 0.05) upregulated for both cell lines exposed to mixed species cultures compared with C. albicans alone. Candida albicans yeast and hyphae were evident in C. albicans only infections. In contrast, monolayers infected by C. albicans, S. mutans, and S. sanguinis exhibited higher microbial invasion with several hyphal aggregates detected. The presence of streptococci in C. albicans infection enhances the virulence and pathogenicity of the fungus with associated increased immune responses and tissue damage. Extrapolation of these findings to oral infection would indicate the added potential benefit of managing bacterial components of biofilms during treatment.

Candida albicans Adhesins Als1 and Hwp1 Modulate Interactions with Streptococcus mutans.

Candida albicans and Streptococcus mutans are known to synergistically interact with each other in the oral cavity. For example, glucosyltransferase B (GtfB), secreted by S. mutans, can bind to the C. albicans cell surface, promoting dual-species biofilm formation. However, the fungal factors mediating interactions with S. mutans are unknown. The C. albicans adhesins Als1, Als3, and Hwp1 are key players in C. albicans single-species biofilm formation, but their roles, if any, in interacting with S. mutans have not been assessed. Here, we investigated the roles of the C. albicans cell wall adhesins Als1, Als3, and Hwp1 on forming dual-species biofilms with S. mutans. We assessed the abilities of the C. albicans wild-type als1Δ/Δ, als3Δ/Δ, als1Δ/Δ/als3Δ/Δ, and hwp1Δ/Δ strains to form dual-species biofilms with S. mutans by measuring optical density, metabolic activity, cell enumeration, biomass, thickness, and architecture of the biofilms. We observed that the C. albicans wild-type strain formed enhanced dual-species biofilms in the presence of S. mutans in these different biofilm assays, confirming that C. albicans and S. mutans synergistically interact in the context of biofilms. Our results reveal that C. albicans Als1 and Hwp1 are major players in interacting with S. mutans, since dual-species biofilm formation was not enhanced when the als1Δ/Δ or hwp1Δ/Δ strains were cultured with S. mutans in dual-species biofilms. Als3, however, does not seem to play a clear role in interacting with S. mutans in dual-species biofilm formation. Overall, our data suggest that the C. albicans adhesins Als1 and Hwp1 function to modulate interactions with S. mutans and could be potential targets for future therapeutics.

Epithelial responses and Candida albicans pathogenicity are enhanced in the presence of oral streptococci

Abstract Experimental models that consider host-pathogen interactions are relevant for improving knowledge about oral candidiasis. The aim of this study was to assess the epithelial immune responses, Candida penetration of cell monolayers, and virulence during mixed species culture infections. Single species cultures of Candida albicans and mixed cultures (C. albicans, Streptococcus mutans, and Streptococcus sanguinis) were used to infect monolayers of HaCaT and FaDu ATCC HTB-43 cells for 12 h. After infection, IL-18 and IL-34 gene expression was measured to assess epithelial cell immune responses, and lactate dehydrogenase (LDH) activity was measured as an indicator of cell damage. Microscopy determined C. albicans morphology and penetration of fungal cells through the keratinocyte monolayer. Monolayers devoid of infection served as controls. Data were analyzed by an ANOVA one-way test followed by Tukey's post-hoc test (α = 0.05). The results found that IL-18 and IL-34 gene expression and LDH activity were significantly (p < 0.05) upregulated for both cell lines exposed to mixed species cultures compared with C. albicans alone. Candida albicans yeast and hyphae were evident in C. albicans only infections. In contrast, monolayers infected by C. albicans, S. mutans, and S. sanguinis exhibited higher microbial invasion with several hyphal aggregates detected. The presence of streptococci in C. albicans infection enhances the virulence and pathogenicity of the fungus with associated increased immune responses and tissue damage. Extrapolation of these findings to oral infection would indicate the added potential benefit of managing bacterial components of biofilms during treatment.

Resumo O objetivo deste estudo foi avaliar a resposta epithelial imune, a colonização da Candida albicans em monocamadas celulares e sua virulência em resposta a infecções de culturas de biofilme multiespécie. Culturas de biofilme monoespécie de C. albicans e culturas mistas (C. albicans, Streptococcus mutans e Streptococcus sanguinis) foram utilizadas para infectar monocamadas de células HaCaT e FaDu por 12 h. Após a infecção, a expressão dos genes IL-18 e IL-34 foi medida para avaliar as respostas imunes das células epiteliais. A atividade da lactato desidrogenase (LDH) foi medida como um indicador de dano celular. A microscopia determinou a morfologia de C. albicans e a penetração das células fúngicas através da monocamada de queratinócitos. Monocamadas em que não houve infecção serviram como controles. Os dados foram analisados por um teste ANOVA one-way seguido pelo teste post-hoc de Tukey (α = 0,05). Os resultados demonstraram que a expressão gênica de IL-18 e IL-34 e a atividade de LDH foram (p < 0,05) reguladas positivamente para ambas as linhagens de células expostas a culturas de espécies mistas em comparação com C. albicans isoladamente. Leveduras de C.albicans e hifas foram evidentes em infecções apenas por C. albicans. Entretanto, monocamadas infectadas por C. albicans, S. mutans e S. sanguinis exibiram maior invasão microbiana com vários agregados de hifas detectados. Dessa maneira, a presença de estreptococos na infecção por C. albicans aumentou a virulência e a patogenicidade do fungo com respostas imunes aumentadas associadas a danos nos tecidos. A extrapolação desses achados para a infecção oral indicaria o potencial benéfico do controle dos componentes bacterianos em biofilmes durante a terapia da candidíase

Demystifying interactions between micro organisms in oral candidiasis biofilm: the key to enhancing treatments

Introduction: candida albicans is a fungal pathogen that can provoke diseases ranging from oral infections to life-threatening systemic disorders. It is now recognized that oral bacteria, such as the genus Streptococcus, establish synergistic relationships with C. albicans, which could potentially increase the fungi's virulence and pathogenicity. Objective: this narrative review aimed to discuss the Candida-Streptococcus mechanisms of interactions and their contribution to increasing oral candidiasis severity. In addition, it provides a background of biofilm formation and potential therapeutical targets. Sources of Data: searches for papers in English were performed in the Pubmed database until May 2022. MeSH and free terms related to the field were used. In vitro studies were selected, tabulated, and qualitative and quantitative data were analyzed descriptively. Synthesis of Data: among the early colonizers bacteria, evidence pointed out that S. gordonnii and S. oralis have major implications in oral candidiasis, in which mixed biofilms increase the infection severity and challenge the host's defense. On the other hand, the outcomes of the interaction between C. albicans and S. mitis, S. sanguinis, or S. mutans remain little explored in the oral candidiasis scenario, albeit evidence pointed out an enhanced fungus population and virulence factors. Conclusion: overall, considering the polymicrobial profile of the infection and the potential to increase Candida-related disease severity, therapeutical strategies should also consider bacteria management.

Introdução: candida albicans é um patógeno fúngico que pode provocar doenças que variam de infecções orais a distúrbios sistêmicos com risco de vida. Hoje se reconhece que as bactérias orais, como o gênero Streptococcus, estabelecem relações sinérgicas com C. albicans, o que pode potencialmente aumentar a virulência e patogenicidade do fungo. Objetivo: esta revisão narrativa teve como objetivo discutir os mecanismos de interação Candida-Streptococcus e sua contribuição para o agravamento da candidíase oral. Além disso, fornece uma breve explanação sobre a formação do biofilme e potenciais alvos terapêuticos. Fonte dos dados: foi realizada pesquisa na base de dados Pubmed para a busca de artigos publicados em Inglês até maio de 2022. Para isso, foram utilizados descritores relacionados ao tema. Estudos in vitro foram selecionados, tabulados e seus resultados quantitativos e qualitativos analisados descritivamente. Síntese dos dados: entre as bactérias denominadas colonizadores iniciais, evidências apontam que S. gordonnii e S. oralis têm implicações importantes na candidíase oral, na qual biofilmes mistos aumentam a gravidade da infecção e desafiam a defesa do hospedeiro. Por outro lado, os desfechos das interações entre C. albicans e S. mitis, S. sanguinis ou S. mutans permanecem pouco explorados no cenário da candidíase oral, apesar de evidências apontarem um aumento dapopulação fúngica e de fatores de virulência. Conclusão: de maneira geral, considerando o perfil polimicrobiano da infecção e o potencial agravamento das doenças provocadas por Candida spp, as estratégias terapêuticas não devem estar focadas apenas no fungo, mas também devem considerar o manejo da bactéria.

Interkingdom interaction between C. albicans and S. salivarius on titanium surfaces.

BACKGROUND: In oral candidiasis models, Candida albicans and Streptococcus salivarius sp. biofilms have an antagonistic relationship. Due to this, S. salivarius have been used experimentally as probiotic. However, the interaction between these microorganisms in the peri-implantitis-like microenvironment remains unknown. This study aimed to evaluate the interaction between C. albicans and S. salivarius biofilms developed on titanium surfaces, under reduced oxygen levels. METHODS: Titanium specimens were pre-conditioned with artificial saliva (1 h, 37 °C). Single-species biofilms of C. albicans (ATCC 90028) and co-culture biofilms of C. albicans and S. salivarius (ATCC 7073) was developed for 24 and 72 h on titanium specimens. Subsequently, the effect of these intervals of biofilm formation and the interactions among the cells were evaluated. Biofilms from cultures were collected and analyzed for cell viability (CFU/mL), biofilm biomass, and total protein dosage. Data were analyzed using Mann-Whitney test (α = 5%). In addition, co-culture biofilms were analyzed using fluorescence microscopy. RESULTS: C. albicans growth did not change due to the presence of S. salivarius. Besides, co-culture biofilms showed a significant difference in the number of viable cells between 24 and 72 h of biofilm development (p < 0.05). The highest biofilm biomass and protein dosage were observed in co-cultures at 72 h of biofilm development. Fluorescence microscopy showed that co-cultures biofilms at 24 h have limited number of pseudo-hyphal and hyphae cells of C. albicans. At 72 h, these types of cells have increased. S. salivarius in both stages of development was present in some clusters surrounded by C. albicans. CONCLUSIONS: Co-cultivation of C. albicans with S. salivarius in biofilms developed on titanium surfaces, under lower oxygen levels, did not affect fungus growth. In addition, S. salivarius did not hind C. albicans virulence. These findings suggest that the use of S. salivarius as a probiotic would be ineffective in peri-implant disease treatment.

Oral candidiasis and denture stomatitis in diabetic patients: Systematic review and meta-analysis.

Here, the prevalence of oral candidiasis and denture stomatitis among diabetic patients compared to healthy ones was summarized through a systematic review with meta-analysis. Medline, Scopus, Web of Science, Lilacs, Cochrane Library, Embase, and the grey literature were searched without restriction, until May 2020. Eligibility criteria were established, data were extracted, and quality assessment was conducted by two trained examiners. Qualitative synthesis was based on the recommendations of Fowkes and Fulton. Two meta-analyses were performed on studies investigating patients with: a) oral candidiasis and b) denture stomatitis. Out of 6034 screened studies, seven were eligible for qualitative and quantitative synthesis; of these, three evaluated oral candidiasis and four evaluated denture stomatitis. Qualitative synthesis showed that the main methodological problems of the studies included sample size, source of controls, matching, and randomization. Diabetic patients had a similar chance of developing oral candidiasis to non-diabetic patients (OR1.40 [0.96; 2.04], p = 0.08, I2 = 94%). However, diabetic patients had a higher chance to present denture stomatitis compared to non-diabetic patients (OR 1.92 [1.42, 2.59] p < 0.0001, I2 = 0%). Therefore, diabetic patients have a higher chance of developing denture stomatitis compared to non-diabetic patients. However, for all analyses, the certainty of the evidence was considered to be very low.

"Inhibitory effect of Brazilian red propolis on Candida biofilms developed on titanium surfaces".

BACKGROUND: Peri-implant inflammation resulting from the presence of Candida biofilms may compromise the longevity of implant-supported dentures. This study evaluated the inhibitory effect of Brazilian red propolis on mono-species biofilms of C. albicans (ATCC 90028) and co-culture biofilms of C. albicans (ATCC 90028) and C. glabrata (ATCC 2001), developed on titanium surfaces. METHODS: Titanium specimens were pre-conditioned with artificial saliva and submitted to biofilm formation (1 × 106 CFU/mL). After 24 h (under microaerophilic conditions at 37 °C) biofilms were submitted to treatment for 10 min, according to the groups: sterile saline solution (growth control), 0.12% chlorhexidine and 3% red propolis extract. Treatments were performed every 24 h for 3 days and analyses were conducted 96 h after initial adhesion. After that, the metabolic activity (MTT assay) (n = 12/group), cell viability (CFU counts) (n = 12/group) and surface roughness (optical profilometry) (n = 6/group) were evaluated. Data from viability and metabolic activity assays were evaluated by ANOVA and Tukey tests. Surface roughness analysis was determined by Kruskal Wallis e Mann Whitney tests. RESULTS: Regarding the mono-species biofilm, the cell viability and the metabolic activity showed that both chlorhexidine and red propolis had inhibitory effects and reduced the metabolism of biofilms, differing statistically from the growth control (p < 0.05). With regards the co-culture biofilms, chlorhexidine had the highest inhibitory effect (p < 0.05). The metabolic activity was reduced by the exposure to chlorhexidine and to red propolis, different from the growth control group (p < 0.05). The surface roughness (Sa parameter) within the mono-species and the co-culture biofilms statistically differed among groups (p < 0.05). CONCLUSIONS: Brazilian red propolis demonstrated potential antifungal activity against Candida biofilms, suggesting it is a feasible alternative for the treatment of peri-implantitis.

Oral candidiasis and denture stomatitis in diabetic patients: Systematic review and meta-analysis

Abstract Here, the prevalence of oral candidiasis and denture stomatitis among diabetic patients compared to healthy ones was summarized through a systematic review with meta-analysis. Medline, Scopus, Web of Science, Lilacs, Cochrane Library, Embase, and the grey literature were searched without restriction, until May 2020. Eligibility criteria were established, data were extracted, and quality assessment was conducted by two trained examiners. Qualitative synthesis was based on the recommendations of Fowkes and Fulton. Two meta-analyses were performed on studies investigating patients with: a) oral candidiasis and b) denture stomatitis. Out of 6034 screened studies, seven were eligible for qualitative and quantitative synthesis; of these, three evaluated oral candidiasis and four evaluated denture stomatitis. Qualitative synthesis showed that the main methodological problems of the studies included sample size, source of controls, matching, and randomization. Diabetic patients had a similar chance of developing oral candidiasis to non-diabetic patients (OR1.40 [0.96; 2.04], p = 0.08, I2 = 94%). However, diabetic patients had a higher chance to present denture stomatitis compared to non-diabetic patients (OR 1.92 [1.42, 2.59] p < 0.0001, I2 = 0%). Therefore, diabetic patients have a higher chance of developing denture stomatitis compared to non-diabetic patients. However, for all analyses, the certainty of the evidence was considered to be very low.