Avaliação in vitro da formação de biofilme por cepas de Candida spp. provenientes de indivíduos diabéticos e não diabéticos em ambiente sem e com suplementação de glicose / In vitro evaluation of biofilm formation by Candida spp. strains from diabetic and non-diabetic individuals in environment without and with glucose supplementation

É notório o papel do diabetes mellitus como fator de risco para o desenvolvimento de doenças bucais, como a candidíase bucal, cárie e a periodontite. Assim, este estudo tem como objetivo avaliar a formação de biofilme por cepas de Candida spp. provenientes de diabéticos e não diabéticos em ambiente sem e com suplementação de glicose. Trata-se de um estudo experimental laboratorial in vitro, em três etapas. Etapas um e dois, de obtenção e identificação de 48 cepas de Candida spp., sendo que 32 de C. albicans e 16 de C. glabrata, com auxílio da técnica de PCR. Ainda, a etapa três, de processamento microbiológico, com a avaliação da capacidade de formação de biofilme por três ensaios distintos: I) determinação do número de unidades formadoras de colônia (UFC/mL); II) matéria seca dos biofilmes; III) taxa de crescimento de biofilme em fundo de placa de poliestireno. Inicialmente, objetivando simular as características observadas in vivo, o fundo das placas de cultivo recebeu 400 µL de saliva humana para formação da película adquirida. Decorrida a incubação a 37 °C por 24 h, a saliva foi descartada e cada poço de cultura recebeu suspensão padronizada das leveduras (106 UFC/mL) em Saubouraud Dextrose Broth sem suplementação e com suplementação de glicose a 2 e 10 mg/mL, e as placas foram incubadas a 37 °C por 48 h. Para avaliação do número de UFC/mL, o biofilme aderido foi coletado, diluído seriamente e cultivado em placas de Petri com Sabouraud Dextrose Agar. Após incubação os resultados foram expressos em log UFC/mL. Para a avaliação da matéria seca, a solução remanescente foi liofilizada e mensurada em balança de precisão. A taxa de crescimento de biofilme foi avaliada por microscopia Operetta CLS High Content e o FilmTracer(TM) LIVE/DEAD Biofilm Viability kit, conforme o protocolo do fabricante. Posteriormente, 10 imagens por poço foram obtidas e digitalizadas com ampliação de 40 ×. A área recoberta por biofilme (µm2) das imagens foi avaliada com auxílio do software Harmony High Content Imaging. Os dados apresentaram distribuição não normal, e a comparação entre as cepas de diabéticos e não diabéticos foi realizada pelo teste U Mann-Whitney. O teste de Kruskal-Wallis one way foi utilizado para verificar diferenças entre as condições de suplementação de glicose. O nível de significância estatística adotado foi de α = 5%. Os valores de UFC/mL mostraram um maior crescimento das cepas de C. albicans dos pacientes diabéticos em relação aos não diabéticos nas três suplementações (p < 0,001). Por outro lado, acerca da matéria seca em 10 mg/mL e da taxa de crescimento de biofilme sem suplementação de glicose e a 2 mg/mL, os resultados indicaram uma formação de biofilme maior para cepas de C. albicans dos não diabéticos (p < 0,001). Em conclusão, cepas de C. albicans e C. glabrata provenientes de diabéticos e não diabéticos em ambiente sem e com suplementação de glicose apresentaram resultados distintos quanto à formação de biofilme, por diferentes técnicas

The role of diabetes mellitus is notorious as a risk factor for development of oral diseases, such as oral candidiasis, dental caries, and periodontitis. Thus, this study aimed to evaluate biofilm formation by Candida spp. strains from diabetic and non-diabetic individuals in environment without and with glucose supplementation. This is an in vitro experimental laboratory study, in three stages. Stages one and two of obtainment and identification of 48 Candida spp. strains, with 32 of C. albicans and 16 of C. glabrata, with the help of PCR technique. Also, stage three, of microbiological processing, with evaluation of biofilm formation capacity by three different assays: I) determination of the number of colony forming units (CFU/mL); II) biofilm dry matter; III) biofilm growth rate on the bottom of polystyrene plates. Initially, aiming to simulate the characteristics observed in vivo, the bottom of the cultivation plates received 400 µL of human saliva for formation of acquired pellicle. After the incubation at 37 °C for 24 h, the saliva was discarded and each culture well received standardized suspension of yeast (106 CFU/mL) in Saubouraud Dextrose Broth without supplementation and with glucose supplementation at 2 and 10 mg/mL, and the plates were incubated at 37 °C for 48 h. To assess the number of CFU/mL, the adhered biofilm was collected, seriously diluted, and cultivated in Petri dishes with Sabouraud Dextrose Agar. After the incubation, the results were expressed in log CFU/mL. To assess the dry matter, the remaining solution was lyophilized and measured on a precision scale. The biofilm growth rate was evaluated by Operetta CLS High Content microscopy and FilmTracer(TM) LIVE/DEAD Biofilm Viability kit, according to manufacturer's protocol. Later, 10 images per well were obtained and digitalized with 40 × magnification. The area covered by biofilm (µm2) of the images was assessed with the help of Harmony High Content Imaging software. Data showed non-normal distribution, and the comparison among the diabetic and non-diabetic strains was performed by Mann-Whitney U test. Kruskal-Wallis one-way test was used to verify differences between conditions of glucose supplementation. The level of statistical significance adopted was α = 5%. The values of CFU/mL showed greater growth of the diabetic patient's strains in relation to the non-diabetic ones (p < 0.001). On the other hand, regarding dry matter at 10 mg/mL and the growth rate of biofilm without glucose supplementation and at 2 mg/mL, the results indicated a higher biofilm formation for strains of C. albicans from non-diabetic individuals (p <0.001). In conclusion, C. albicans and C. glabrata strains from diabetic and non-diabetic individuals in environment without and with glucose supplementation showed different results concerning the biofilm formation, using different techniques

Elimination of an intraradicular biofilm composed of enterococcus faecalis and candida albicans using a negative apical pressure irrigation system / Eliminación de una biopelícula intraradicular compuesta por enterococcus faecalis y candida albicans utilizando un sistema de irrigación de presión apical negativa

Endodontic treatment consists of the cleaning and disinfecting the root canal system, which is achieved using adequate mechanical instru-mentation and chemical irrigation. Endodontic microorganisms are present in root canals in the form of a biofilm, and their elimination ensures the success of endodontic treatment. Irrigation is a key factor contributing to the elimination of this intraconduct biofilm, and different irrigator agents and irrigation techniques, such as irrigation with negative apical pressure, a novel automated irrigation mechanism based on suction intraconduct, have been used. In this study, we evaluated the ability of a negative apical pressure system with different concentrations of sodium hypochlorite and durations to reduce the microbial load. Materials and Methods: An intraradicular biofilm composed of Enterococcus faecalis and Candida albicans was generated during twenty-one days of static culture on one hundred mesio-vestibular roots of upper molars with complex curvatures greater than 30°C, and the roots were classified in six groups with different concentrations and contact times of sodium hypochlorite. Subsequently, the reduction in the microbial load was measured with McFarland scale and the enumeration of colony forming units and was evaluated with scanning electronic microscopy. Results: We observed a significant difference in the reduction of the microbial load prior to instrumentation compared with postinstrumentation between the groups treated with 2.25% and 5.25% NaOCl for 30, 60 and 90 seconds of contact time (p<0.05), but we did not observe differences in the reduction of microbial load between different contact times and concentrations of sodium hypochlorite employed (p>0.05). Conclusion: Negative apical pressure is a good option for irrigation in endodontics, as it allows the passage of the irrigation fluid along the total length of the root canal and produces a better antimicrobial effect.

El tratamiento de endodoncia consiste en la limpieza y desinfección del sistema de conducto radicular, lo que se logra utilizando instrumentación mecánica adecuada y riego químico. Los microorganismos endodónticos están presentes en los conductos radiculares en forma de una biopelícula, y su eliminación asegura el éxito del tratamiento endodóntico. La irrigación es un factor clave que contribuye a la eliminación de esta biopelícula intraconductora, y se han utilizado diferentes agentes irrigadores y técnicas de irrigación, como la irrigación con presión apical negativa, un nuevo mecanismo automatizado de irrigación basado en la intraconducción de succión. En este estudio, evaluamos la capacidad de un sistema de presión apical negativa con diferentes concentraciones de hipoclorito de sodio y duraciones para reducir la carga microbiana. Material y Métodos: Se generó una biopelícula intraradicular compuesta por Enterococcus faecalisy Candida albicans durante veintiún días de cultivo estático en cien raíces mesio-vestibulares de molares superiores con curvaturas complejas superiores a 30°C, y las raíces se clasificaron en seis grupos con diferentes concentraciones y tiempos de contacto de hipoclorito de sodio. Posteriormente, la reducción en la carga microbiana se midió con la escala de McFarland y la enumeración de las unidades formadoras de colonias y se evaluó con microscopía electrónica de barrido. Resultado: Observamos una diferencia significativa en la reducción de la carga microbiana antes de la instrumentación en comparación con la postinstrumentación entre los grupos tratados con NaOCl 2.25% y 5.25% durante 30, 60 y 90 segundos de tiempo de contacto (p<0.05), pero lo hicimos No se observan diferencias en la reducción de la carga microbiana entre los diferentes tiempos de contacto y las concentraciones de hipoclorito de sodio empleado (p>0.05). Conclusión:La presión apical negativa es una buena opción para el riego en endodoncia, ya que permite el paso del líquido de riego a lo largo de todo el conducto radicular y produce un mejor efecto antimicrobiano.

Role of Candida albicans on enamel demineralization and on acidogenic potential of Streptococcus mutans in vitro biofilms

Abstract There is growing evidence that C. albicans is associated with dental caries, but its role on caries development needs to be better clarified. Objective: To evaluate at the hard tissue level the effect of C. albicans on the cariogenic potential of S. mutans biofilms focusing on the mineral profile of induced carious lesions. This study also aimed to evaluate the effect of C. albicans on the acidogenic potential of S. mutans biofilms. Methodology: Dual-species (CA+SM) and single-species biofilms (CA or SM) were grown on the surface of enamel slabs in the presence of glucose/sucrose supplemented culture medium for 24, 48 and 72 hours. Demineralization was evaluated through percentage of surface microhardness change (%SMC) and transversal microradiography analysis (ILM and LD) and pH of the spent medium was recorded daily. Data were analyzed by two-way ANOVA followed by Bonferroni correction. Results: %SMC was statistically different among the biofilms at each time point being the highest for SM biofilms and the lowest for CA biofilms which also differed from CA+SM biofilms [SM (24 h: 47.0±7.3; 48 h: 66.3±8.3; 72 h: 75.4±3.9); CA (24 h: 7.3±3.3; 48 h: 7.1±6.4; 72 h: 6.6±3.6); CA+SM (24 h: 35.9±7.39.1; 48 h: 47.2±9.5; 72 h: 47.6±9.5)]. pH of spent medium was statistically lower for SM biofilms compared to the other biofilms at each time point and remained constant over time while pH values increased from 24 to 72 h for both CA and CA+SM biofilms [SM (24 h: 4.4±0.1; 48 h: 4.4±0.1; 72 h: 4.5±0.1); CA (24 h: 6.9±0.3; 48 h: 7.2±0.2; 72 h: 7.5±0.2); CA+MS (24 h: 4.7±0.2; 48 h: 5.1±0.1; 72 h: 6.1±0.6)]. IML and LD for SM biofilms increased over time while no difference was observed from 24 to 72 h for the other biofilms. Conclusions: The present data suggest that C. albicans has low enamel demineralization potential and the presence of C. albicans can reduce both the cariogenic and acidogenic potentials of S. mutans biofilms.

Culture media profoundly affect Candida albicans and Candida tropicalis growth, adhesion and biofilm development

As there are sparse data on the impact of growth media on the phenomenon of biofilm development for Candida we evaluated the efficacy of three culture media on growth, adhesion and biofilm formation of two pathogenic yeasts, Candida albicans and Candida tropicalis. The planktonic phase yeast growth, either as monocultures or mixed cultures, in sabouraud dextrose broth (SDB), yeast nitrogen base (YNB), and RPMI 1640 was compared, and adhesion as well as biofilm formation were monitored using MTT and crystal violet (CV) assays and scanning electron microscopy. Planktonic cells of C. albicans, C. tropicalis and their 1:1 co-culture showed maximal growth in SDB. C. albicans/C. tropicalis adhesion was significantly facilitated in RPMI 1640 although the YNB elicited the maximum growth for C. tropicalis. Similarly, the biofilm growth was uniformly higher for both species in RPMI 1640, and C. tropicalis was the slower biofilm former in all three media. Scanning electron microscopy images tended to confirm the results of MTT and CV assay. Taken together, our data indicate that researchers should pay heed to the choice of laboratory culture media when comparing relative planktonic/biofilm growth of Candida. There is also a need for standardisation of biofilm development media so as to facilitate cross comparisons between laboratories.

Mixed biofilms formed by C. albicans and non-albicans species: a study of microbial interactions

Abstract Most Candida infections are related to microbial biofilms often formed by the association of different species. The objective of this study was to evaluate the interactions between Candida albicans and non-albicans species in biofilms formed in vitro. The non-albicans species studied were:Candida tropicalis, Candida glabrata andCandida krusei. Single and mixed biofilms (formed by clinical isolates of C. albicans and non-albicans species) were developed from standardized suspensions of each strain (107 cells/mL), on flat-bottom 96-well microtiter plates for 48 hour. These biofilms were analyzed by counting colony-forming units (CFU/mL) in Candida HiChrome agar and by determining cell viability, using the XTT 2,3-bis (2-methoxy-4-nitro-5-sulphophenyl)-5-[(phenylamino) carbonyl]-2H-tetrazolium hydroxide colorimetric assay. The results for both the CFU/mL count and the XTT colorimetric assay showed that all the species studied were capable of forming high levels of in vitro biofilm. The number of CFU/mL and the metabolic activity of C. albicans were reduced in mixed biofilms with non-albicans species, as compared with a singleC. albicans biofilm. Among the species tested, C. krusei exerted the highest inhibitory action against C. albicans. In conclusion, C. albicans established antagonistic interactions with non-albicans Candida species in mixed biofilms.

Evaluation of Swallowing in Infants with Congenital Heart Defect

Introduction Surgical repair of congenital heart disease in the first years of life compromises the coordination of the suction, breathing, and swallowing functions. Objective To describe the alterations in swallowing found in infants with congenital heart defect during their hospitalization. Methods Prospective, cross-sectional study in a reference hospital for heart disease. The sample consisted of 19 postsurgical patients who underwent an evaluation of swallowing. The infants included were younger than 7 months and had a diagnosis of congenital heart defect and suspected swallowing difficulties. Results Of the 19 infants with congenital heart defect, the median age was 3.2 months. A significant association was found between suction rhythm and dysphagia (p = 0.036) and between oral-motor oral feeding readiness and dysphagia (p = 0.014). Conclusions The data suggest that dysphagia often occurs after surgery in infants with congenital heart defect. Infants with congenital heart defect had very similar behavior to preterm infants in terms of oral feeding readiness. .

Evaluación del cultivo liofilizado de Candida albicans utilizado en esquemas de certificaciones de calidad / Evaluation of the lyophilized culture of Candida albicans used in quality certification schemes

Introducción: el laboratorio de control microbiológico de la UEB Laboratorios Liorad dispone de una colección de cultivos microbianos para la conservación de microorganismos, donde se encuentra depositada la levadura Candida albicans que se emplea en esquemas de certificaciones de calidad establecidos para la evaluación de ensayos como: promoción de crecimiento de los medios de cultivos, validación de técnicas microbiológicas, entre otros. Objetivo: evaluar los resultados de la conservación de esta cepa por el método de liofilización durante un periodo de ocho años. Métodos: para el crecimiento de la cepa se utilizó el medio de cultivo Caldo Saboraud y variantes de sustancias lioprotectoras puras como: (leche descremada al 20 por ciento, glicerol 20 por ciento, sacarosa al 10 por ciento y peptona 5 por ciento) así como la mezcla de lioprotectores (leche 10 por ciento, sacarosa 5 por ciento, glicerol 5 por ciento). Se evaluó viabilidad, pureza y estabilidad genética de esta cepa durante el tiempo objeto de estudio. Resultados: las características propias de la especie estudiada se mantuvieron inalterables con un elevado grado de pureza en todas las variantes estudiadas. En cuanto a la supervivencia, cuando se emplearon las sustancias lioprotectoras puras se evidenció una marcada disminución de la viabilidad. No así al emplear la mezcla de lioprotectores que mantuvo niveles de viabilidad por encima del límite establecido durante todo el tiempo objeto de estudio. Conclusiones: los valores obtenidos en cuanto a la supervivencia de este microorganismo permiten inferir que para la conservación por largos periodos de tiempo la variante donde se empleó mezclas de lioprotectores resultó una buena opción para la conservación de C. albicans(AU)

Introduction: the microbiological control laboratory at the Basic Enterprise Unit Liorad Laboratories stores a collection of microbial cultures for the preservation of microorganisms, including the Candida albicans yeast used in the quality certification schemes established for the evaluation of assays such as fostering of culture medium growth and validation of microbiological techniques, among others. Objective: evaluate the results obtained in the preservation of this strain by the lyophilization method during a period of eight years. Methods: for strain growth, use was made of Saboraud broth culture medium as well as variants of pure lyoprotective substances such as 20 percent skimmed milk, 20 percent glycerol, 10 percent saccharose and 5 percent peptone, and the mixture of lyoprotectors (10 percent milk, 5 percent saccharose, 5 percent glycerol). An evaluation was conducted of the viability, purity and genetic stability of the strain during the study period. Results: characteristics typical of the study species remained unchanged with a high degree of purity in all the variants examined. As to survival, a marked reduction in viability was observed when pure lyoprotective substances were used, but not with the mixture of lyoprotectors, in which case viability levels exceeded the established limit during the entire study period. Conclusions: the survival values obtained for this microorganism indicate that preservation for long periods with mixtures of lyoprotectors was a good option for the preservation of C. albicans(AU)

Stenotrophomonas maltophilia interferes via the DSF-mediated quorum sensing system with Candida albicans filamentation and its planktonic and biofilm modes of growth / Stenotrophomonas maltophilia interfiere a través del sistema de "quorum sensing" mediado por DSF con la filamentación y el crecimiento planctónico y en biofilm de Candida albicans

Stenotrophomonas maltophilia is a nosocomial pathogen of increasing importance. S. maltophilia K279a genome encodes a diffusible signal factor (DSF) dependent quorum sensing (QS) system that was first identified in Xanthomonas campestris pv. campestris. DSF from X. campestris is a homologue of farnesoic acid, a Candida albicans QS signal which inhibits the yeast-to-hyphal shift. Here we describe the antagonistic effects of S. maltophilia on C. albicans on filamentation as well as on its planktonic and biofilm modes of growth. To determine the role of the DSF-mediated quorum sensing system in these effects, C. albicans ATCC 10231 and C. albicans tup1 mutant, locked in the filamentous form, were grown with K279a or with its rpfF deletion mutant (DSF-). A significant reduction in viable counts of C. albicans was observed in planktonic cocultures with K279a as well as in mixed biofilms. Furthermore, no viable cells of C. albicans tup1 were recovered from K279a mixed biofilms. Fungal viability was also assessed by labeling biofilms with SYTO 9 and propidium iodide. Confocal images showed that K279a can kill hyphae and also yeast cells. Light microscopic analysis showed that K279a severely affects hyphae integrity. On the other hand, the presence of K279a rpfF did not affect fungal morphology or viability. In conclusion, we report for the first time that S. maltophilia interferes with two key virulence factors of C. albicans, the yeast-to-hyphal transition and biofilm formation. DSF could be directly responsible for these effects or may induce the gene expression involved in antifungal activity.

Stenotrophomonas maltophilia es un patógeno nosocomial de importancia creciente. El genoma de S. maltophilia K279a codifica un factor de señalización difusible (DSF), autoinductor de "quorum sensing" (QS), identificado previamente en Xanthomonas campestris pv. campestris. El DSF de X. campestris es homólogo del ácido farnesoico, señal de QS de Candida albicans, que inhibe la transición levadura-hifa. En este trabajo se describe el efecto antagónico de S. maltophilia sobre la filamentación y el crecimiento planctónico y en biofilms de C. albicans. Para determinar la participación del sistema de QS mediado por el DSF en dichos efectos, C. albicans ATCC 10231 y la mutante C. albicans tup1, que solo crece en forma filamentosa, fueron cultivadas en presencia de K279a o de su mutante K279a rpfF (DSF-). Se observó una reducción significativa del número de viables de C. albicans en cultivos planctónicos y biofilms desarrollados en presencia de K279a. Es de señalar que no se recuperaron células viables de C. albicans tup1 a partir de biofilms mixtos en presencia de K279a. Las imágenes de microscopía confocal revelaron que K279a produce la muerte de hifas y levaduras en biofilms mixtos teñidos con ioduro de propidio y SYTO 9. El análisis por microscopía óptica mostró que K279a afecta la integridad de las hifas. En cambio, la presencia de K279a rpfF no afectó la morfología ni la viabilidad fúngica. En conclusión, informamos por primera vez que S. maltophilia interfiere con dos factores de virulencia de C. albicans, la transición levadura-hifa y la formación de biofilms. Estos efectos pueden ser mediados por el DSF en forma directa o a través de la inducción de genes involucrados en la actividad antifúngica.

Candida albicans biofilms and MMA surface treatment influence the adhesion of soft denture liners to PMMA resin

The effect of Candida albicans biofilms and methyl methacrylate (MMA) pretreatment on the bond strength between soft denture liners and polymethyl methacrylate (PMMA) resin was analyzed. Specimens were prepared and randomly divided with respect to PMMA pretreatment, soft liner type (silicone-based or PMMA-based), and presence or absence of a C. albicans biofilm. Samples were composed of a soft denture liner bonded between two PMMA bars. Specimens (n = 10) were incubated to produce a C. albicans biofilm or stored in sterile PBS for 12 days. The tensile bond strength test was performed and failure type was determined using a stereomicroscope. Surface roughness (SR) and scanning electron microscopy (SEM) analysis were performed on denture liners (n = 8). Highest bond strength was observed in samples containing a silicone-based soft liner and stored in PBS, regardless of pretreatment (p < 0.01). Silicone-based specimens mostly underwent adhesive failures, while samples containing PMMA-based liners predominantly underwent cohesive failures. The silicone-based specimens SR decreased after 12 days of biofilm accumulation or PBS storage, while the SR of PMMA-based soft liners increased (p < 0.01). The PMMA-based soft liners surfaces presented sharp valleys and depressions, while silicone-based specimens surfaces exhibited more gentle features. In vitro exposure to C. albicans biofilms reduced the adhesion of denture liners to PMMA resin, and MMA pretreatment is recommended during relining procedures.

Effect of alcohols on filamentation, growth, viability and biofilm development in Candida albicans

In this study we report the potential of alcohols as morphogenetic regulators in Candida albicans. All the alcohols tested influenced various modes of growth like planktonic as well as biofilm forms. Viability was affected at high concentrations. Among the alcohols, the response of C. albicans to amyl alcohol (pentanol) was noteworthy. Amyl alcohol at a concentration 0.5% which was not inhibitory to growth and viability specifically inhibited morphogenetic switching from yeast to hyphal forms. It also inhibited normal biofilm development favoring yeast dominated biofilms. Based on this study we hypothesize that alcohols produced under anaerobic conditions may not favor biofilm development and support dissemination of yeast cells. Since anaerobic conditions are not found to favor production of quorum sensing molecules like farnesol, the alcohols may play a role in morphogenetic regulation.

Chloroquine sensitizes biofilms of Candida albicans to antifungal azoles

Biofilms formed by Candida albicans, a human pathogen, are known to be resistant to different antifungal agents. Novel strategies to combat the biofilm associated Candida infections like multiple drug therapy are being explored. In this study, potential of chloroquine to be a partner drug in combination with four antifungal agents, namely fluconazole, voriconazole, amphotericin B, and caspofungin, was explored against biofilms of C. albicans. Activity of various concentrations of chloroquine in combination with a particular antifungal drug was analyzed in a checkerboard format. Growth of biofilm in presence of drugs was analyzed by XTT-assay, in terms of relative metabolic activity compared to that of drug free control. Results obtained by XTT-metabolic assay were confirmed by scanning electron microscopy. The interactions between chloroquine and four antifungal drugs were determined by calculating fractional inhibitory concentration indices. Azole resistance in biofilms was reverted significantly (p < 0.05) in presence of 250 µg/mL of chloroquine, which resulted in inhibition of biofilms at very low concentrations of antifungal drugs. No significant alteration in the sensitivity of biofilms to caspofungin and amphotericin B was evident in combination with chloroquine. This study for the first time indicates that chloroquine potentiates anti-biofilm activity of fluconazole and voriconazole.

Effectiveness of mechanical brushing with different denture cleansing agents in reducing in vitro Candida albicans biofilm viability

The adhesion of Candida albicans to surfaces is the prerequisite for occurrence of denture stomatitis, a common disease diagnosed among denture wearers. A routine of denture cleansing is essential to prevent biofilm formation and the onset of this infection. The aim of this study was to investigate the effectiveness of combining brushing and cleansing agents in killing C. albicans biofilm. Disks of acrylic resin were made, sterilized, and inoculated with C. albicans (10(7) cfu/mL). After incubation (37°C/48 h), specimens were randomly assigned to 10 experimental groups (n=9): 5 subjected to brushing with distilled water or cleansing agents - dentifrice slurry, 2% chlorhexidine gluconate (CHX), 1% sodium hypochlorite (NaOCl), and Polident fresh cleanse® (combined method) - and 4 exposed to the cleansing agents without brushing (immersion). Non-cleansed specimens were used as positive controls. The viability of cells was evaluated by XTT reduction method. Results were analyzed by Mann-Whitney and Kruskal-Wallis tests (α=0.05). The combined method was significantly more effective (p<0.0001) in reducing biofilm viability than the immersion. Brushing with CHX and NaOCl resulted in 100% removal of the biofilm. Immersion in the agents reduced significantly (p<0.0001) the biofilm viability, with CHX being the most effective (p<0.0001). The use of the combined method of brushing with cleansing agents is an effective method to reduce C. albicans biofilm, being CHX and NaOCl the most effective solutions.

A adesão de Candida albicans às superfícies é o primeiro passo para o desenvolvimento da estomatite protética, uma infecção frequente diagnosticada entre os usuários de próteses. Uma adequada higienização é essencial para prevenir a formação de biofilme microbiano e o início desta infecção. O objetivo deste estudo foi avaliar a efetividade da escovação com diferentes soluções na eliminação de biofilme de C. albicans. Para isso, discos de resina acrílica foram confeccionados, esterilizados e inoculados com uma suspensão de 10(7) células/mL de C. albicans. Após incubação (37°C/48 h), os espécimes foram aleatoriamente divididos em 10 grupos experimentais (n=9): 5 submetidos à escovação com água ou agentes de limpeza (água destilada, dentifrício, digluconato de clorexidina (CHX) a 2%, hipoclorito de sódio (NaOCl) a 1% e Polident fresh cleanse®) e 4 apenas imersos nos agentes de limpeza. Espécimes não submetidos à higienização foram utilizados como controle positivo. A viabilidade celular foi verificada pelo teste de redução do XTT. Os resultados obtidos foram analisados pelos testes de Mann-Whitney e Kruskal-Wallis (α=0,05). A escovação com todos os agentes de limpeza apresentou redução significativamente superior (p<0,0001) na viabilidade do biofilme quando comparada à exposição dos espécimes às soluções. Escovação com CHX a 2% e NaOCl a 1% resultaram em 100% de inativação do biofilme. A exposição aos agentes de limpeza resultou em redução significativa (p<0,0001) na viabilidade celular, com CHX a 2% sendo o mais efetivo (p<0,0001). A utilização de agentes de limpeza em associação ao método de escovação provou ser efetivo para reduzir biofilme C. albicans, sendo as soluções de CHX e NaOCl as mais efetivas.

Multi-species biofilm of Candida albicans and non-Candida albicans Candida species on acrylic substrate

OBJECTIVE: In polymicrobial biofilms bacteria extensively interact with Candida species, but the interaction among the different species of the Candida is yet to be completely evaluated. In the present study, the difference in biofilm formation ability of clinical isolates of four species of Candida in both single-species and multi-species combinations on the surface of dental acrylic resin strips was evaluated. MATERIAL AND METHODS: The species of Candida, isolated from multiple species oral candidiasis of the neutropenic patients, were used for the experiment. Organisms were cultured on Sabouraud dextrose broth with 8 percent glucose (SDB). Biofilm production on the acrylic resins strips was determined by crystal violet assay. Student's t-test and ANOVA were used to compare in vitro biofilm formation for the individual species of Candida and its different multi-species combinations. RESULTS: In the present study, differences between the mean values of the biofilm-forming ability of individual species (C. glabrata>C. krusei>C. tropicalis>C. albicans) and in its multi-species' combinations (the highest for C. albicans with C. glabrata and the lowest for all the four species combination) were reported. CONCLUSIONS: The findings of this study showed that biofilm-forming ability was found greater for non-Candida albicans Candida species (NCAC) than for C. albicans species with intra-species variation. Presence of C. albicans in multi-species biofilms increased, whereas; C. tropicalis decreased the biofilm production with all other NCAC species.

Influence of artificial saliva in biofilm formation of Candida albicans in vitro

Due to the increase in life expectancy, new treatments have emerged which, although palliative, provide individuals with a better quality of life. Artificial saliva is a solution that contains substances that moisten a dry mouth, thus mimicking the role of saliva in lubricating the oral cavity and controlling the existing normal oral microbiota. This study aimed to assess the influence of commercially available artificial saliva on biofilm formation by Candida albicans. Artificial saliva I consists of carboxymethylcellulose, while artificial saliva II is composed of glucose oxidase, lactoferrin, lysozyme and lactoperoxidase. A control group used sterile distilled water. Microorganisms from the oral cavity were transferred to Sabouraud Dextrose Agar and incubated at 37°C for 24 hours. Colonies of Candida albicans were suspended in a sterile solution of NaCl 0.9%, and standardisation of the suspension to 106 cells/mL was achieved. The acrylic discs, immersed in artificial saliva and sterile distilled water, were placed in a 24-well plate containing 2 mL of Sabouraud Dextrose Broth plus 5% sucrose and 0.1 mL aliquot of the Candida albicans suspension. The plates were incubated at 37°C for 5 days, the discs were washed in 2 mL of 0.9% NaCl and placed into a tube containing 10 mL of 0.9% NaCl. After decimal dilutions, aliquots of 0.1 mL were seeded on Sabouraud Dextrose Agar and incubated at 37°C for 48 hours. Counts were reported as CFU/mL (Log10). A statistically significant reduction of 29.89% (1.45 CFU/mL) of Candida albicans was observed in saliva I when compared to saliva II (p = 0.002, considering p≤0.05).

In vitro adherence of Candida albicans isolated from patients with chronic periodontitis

Adherence is considered an extremely important virulence factor in yeast. OBJECTIVE: The aim of this study was to analyze the adherence to epithelial cells of C. albicans isolated from patients with chronic periodontitis in comparison to healthy patients. MATERIAL AND METHODS: Candida albicans cells isolated from individuals with chronic periodontitis (n=25) and healthy controls (n=25) were included in this study. Suspensions of C. albicans (10(6) cells/mL) and epithelial cells (10(5) cells/mL) were mixed and incubated at 37ºC for 1 h. The number of yeasts adhered to 25 epithelial cells was counted. RESULTS: The number of C. albicans cells adhered to epithelial cells was statistically higher in the chronic periodontitis group than in the control group (Student's t-test, p=0.000). CONCLUSION:The results of the present study suggest a higher Candida adherence of samples isolated from patients with chronic periodontitis.

Antimicrobial effects of boesenbergia pandurata and piper sarmentosum leaf extracts on planktonic cells and biofilm of oral pathogens

Increasing awareness of hazards associated with the use of antibiotic and chemical agents has accelerated investigations into plants and their extracts as new sources of antimicrobial agents. Therefore, this study aimed at determining the effects of oil and 95% ethanol extracts of Boesenbergia pandurata rhizomes and Piper sarmentosum leaf against four oral pathogens which were Streptococcus mutans, Lactobacillus sp., Aggregatibacter actinomycetemcomitans and Candida albicans. Employing the disc diffusion and broth microdilution methods, the results showed that B. pandurata oil [BPO] was the most effective extract against C albicans. Time-kill assay with the BPO demonstrated killing of C. albicans at concentrations equal to 2 and 2.5 times the MIC. The times required to reach the fungicidal endpoint at 2 and 2.5 times the MIC were 60 and 44 min, respectively. In addition, our results also demonstrated that the BPO possesses potent anti- Candida biofilm activity in vitro. Therefore, the BPO could be considered as a natural antifungal agent against Candida infections and has significant potential for further investigation

Frequencia dos agentes etiologicos de onicomicose em pacientes hansenianos / ?

A anestesia de extremidades e uma caracteristica da hanseniase; os danos neural e vascular levam a deformidade. Uma das consequencias comuns e o comprometimento ungueal, apresentando-se principalmente sob a forma de distrofia, que pode ser colonizada por fungos. A infeccao micotica facilita o aparecimento de infeccoes bacterianas secundarias, que podem desencadear reacoes hansenicas, comprometendo a qualidade de vida do paciente. Neste trabalho, estudou-se a frequencia de agentes de onicomicose em 104 pacientes hansenianos com lesoes clinicamente compativeis. Foram utilizados exame micologico direto e cultura, que foi repetida por tes vezes. Encontrou-se frequencia de 91,3 por cento de onicomicose, com predominio de leveduras (60,0 por cento). Houve discordancia entre exame micologico direto e cultura em 27,6 por cento dos casos. Em 21,6 por cento dos exames diretos microscopicamente positivos nao foi observado crescimento fungico na respectiva cultura e em 6 por cento dos casos verificou-se exame micologico direto negativo e cultura positiva. Os dermatofitos represemtam 26, 7 por cento dos isolamentos, sendo Trichophyton rubrum o mais frequente (15, 3 por cento). Os fungos filamentosos nao dermatofitos foram 13,3 por cento das cepas isoladas, com maior frequencia do genero Scytalidium (10,5 por cento). Candida parapsilosis foi a especie de levedura mais frequente (21,53) por cento), seguida de C. tropicalis (7,69 por cento) e C. albicans (7,69 por cento). Os achados surgerm que as unhas destes pacientes sao suscetiveis as onicomicoses, principalmente com predisposicao a infeccao por agentes oportunistas...

Resistence of Candida species to antifungal agents: molecular mechanisms and clinical consequences

Candida albicans and related species pathogenic for man become resistent to antifungal agents, in particular triazole compounds, by expression of efflux pumps that reduce drug accumulation, alteration of the structure or concentration of antifungal target proteins, and alteration of membrane sterol composition. The clinical consequences of antifungal resistence can be seen in treatment failures in patients and in changes in the prevalence of Candida species causing disease. These effects were seen unequivocally in HIV-infected patients with oropharyngel candida infections, but their incidence has decreased dramatically with the introduction of highly active antiretroviral therapy. The evidence for similar emergence of antifungal-resistant yeast strains and species in other types of candida infections is confounded by non-standardised susceptibillity testing methods and definitions of a resistent fungal isolate. Recent large-scale surveys of yeasts isolated from blood cultures, based on standardised methodology and resistence definitions, do not support the view that antifungal resistance in pathogenic yeasts contitutes a significant or growing therapeutic problem

The presumptive identification of Candida albicans with germ tube induced by high temperature

For direct identification of Candida albicans from other Candida species, the chlamydospore formation and the mycelial transition induced by high temperature and by sera were examined in 198 Candida isolates. The germ tubes of C. albicans developed early at 30 min in high temperature-induction, but at 60 min in serum-induction. C. albicans generated germ tubes well at concentrations lower than 2 x 10(7) cells/ml, but the germ tube formation was markedly restrained at concentrations higher than 4 x 10(7) cells/ml. In a serum-free, yeast extract-peptone-dextrose (YEPD) medium, C. albicans grew as a yeast form at 30 degrees C and as a mycelial form at 35-42 degrees C. Mycelial development was maximal at 37 degrees C in serum and at 39 degrees C in YEPD. Germ tubes were formed within 30 min in YEPD at 39 degrees C, but after 60 min in serum at 37 degrees C. Our examination showed that the 39 degrees C-induced germ tube formation tests were very reliable (sensitivity 100%, specificity 100%) at discerning C. albicans from other Candida species. These results suggest that the high temperature-induced germ tube formation testing could be a useful identification method of C. albicans in clinical laboratories.

Inhibition of macrophage phagocytosis of Escherichia coli by mannose and mannan

1. We investigated the possibility that Candida albicans and Escherichia coli are interiorized by thioglycollate-elicited peritoneal macrophages by interacting with the same receptor. 2. D-mannose (50 mM), a sugar recognized by mannose receptors, reduced the phatfocytosis of C. albicans and E. coli to 46% and 38% of control values, respectively. The presence of 50 mM galactose did not affect the phagocytosis of either microorganism. However, mannan from saccharomyces cerevisiae (0.5 mg/ml) inhibited phagocytosis by 70% for both microorganisms. 3. The ingestion of C. albicans and E. coli was reduced by 85% and 95%, respectively, in the presence of 10 mM EGTA. 4. These results suggest that the mannose receptor, which mediates the recognition of C. albicans by macrophages, might also mediate the phagocytosis of Escherichia coli 0111