Evaluation of biofilm formation on acrylic resin surfaces coated with silicon dioxide: an in situ study.

Biofilm on acrylic resin dental prostheses may cause gingival inflammation. This study evaluated the influence of a silicon dioxide coating layer applied onto acrylic resin on the adhesion of microorganisms. Blocks (5 x 5 x 3 mm) of acrylic resin were evaluated for surface roughness and divided into two groups: control (CG) and coated with silicon dioxide (LG group). The specimens were evaluated by scanning electron microscopy (n = 1) and by contact angle analysis (n = 3). For the in situ study, 20 volunteers wore acrylic palatal devices containing three samples from each group (n = 60) for 2 days. The biofilm formed was quantified by metabolic activity and total biomass using the crystal violet assay. The results were subjected to Bartlett's normality test and Gamma model with random effect for the response variable (α = 5%). The mean contact angle of the coated group was significantly lower than that of the uncoated group (p < 0.05). The metabolic activity of microorganisms in the biofilm on the blocks treated with coating was significantly lower than that of control blocks (p = 0.02). Regarding the amount of extracellular matrix produced by the microorganisms, there was no difference between the CG and LG group (p = 0.05). The application of a silicon dioxide coating on acrylic resin reduced the activity of the polymicrobial biofilm formed in situ. This coating may be advantageous for patients with conventional complete dentures or implants made of acrylic resin and who have motor difficulties that prevent them from cleaning their prostheses properly.

Rhodotorula sp. and Trichosporon sp. are more Virulent After a Mixed Biofilm.

Rhodotorula spp. and Trichosporon spp. are opportunistic pathogens, and although an association between these two species in the same infection appears to be uncommon, it has been reported. This is the first study that aimed to evaluate the pathogenesis of a co-infection by R. mucilaginosa and T. asahii, using a new in vivo model, the Zophobas morio larvae. Suspensions from planktonic and biofilm-recovered cells were injected in the larvae as in monospecies as mixed (a ratio of 1:1 for both agents of a of 105 inoculum). Individual and mixed biofilms of R. mucilaginosa and T. asahii were produced for 24 and 48 h, and they were partially characterized by crystal violet and reduction of tetrazolium salt. When evaluating the impact of the planktonic suspension in vivo we verified that the fungi in monoculture were more able to kill the larvae than those from planktonic mixed suspension. On the other hand, regarding biofilm-recovered cells, there was an increase in the death of larvae infected for mixed suspensions. Moreover, the death rate was more pronounced when the larvae were infected with 48 h biofilm-recovered cells than the 24 h ones. T. asahii was the best producer of total biomass, mainly in 48 h. The metabolic activity for both yeasts organized in biofilm maintained the same pattern between 24 and 48 h. The present study proves a synergistic interaction between R. mucilaginosa and T. asahii after an experience in a mixed biofilm. Our results suggest that both species were benefited from this interaction, acquiring a greater potential for virulence after passing through the biofilm and this ability was acquired by the cells released from the biofilm.

Evaluation of biofilm formation on acrylic resin surfaces coated with silicon dioxide: an in situ study

Abstract Biofilm on acrylic resin dental prostheses may cause gingival inflammation. This study evaluated the influence of a silicon dioxide coating layer applied onto acrylic resin on the adhesion of microorganisms. Blocks (5 x 5 x 3 mm) of acrylic resin were evaluated for surface roughness and divided into two groups: control (CG) and coated with silicon dioxide (LG group). The specimens were evaluated by scanning electron microscopy (n = 1) and by contact angle analysis (n = 3). For the in situ study, 20 volunteers wore acrylic palatal devices containing three samples from each group (n = 60) for 2 days. The biofilm formed was quantified by metabolic activity and total biomass using the crystal violet assay. The results were subjected to Bartlett's normality test and Gamma model with random effect for the response variable (α = 5%). The mean contact angle of the coated group was significantly lower than that of the uncoated group (p < 0.05). The metabolic activity of microorganisms in the biofilm on the blocks treated with coating was significantly lower than that of control blocks (p = 0.02). Regarding the amount of extracellular matrix produced by the microorganisms, there was no difference between the CG and LG group (p = 0.05). The application of a silicon dioxide coating on acrylic resin reduced the activity of the polymicrobial biofilm formed in situ. This coating may be advantageous for patients with conventional complete dentures or implants made of acrylic resin and who have motor difficulties that prevent them from cleaning their prostheses properly.

Standardization of resazurin use in susceptibility testing of natural products against yeasts in planktonic cells and in biofilms formation

Natural products, such as the ethanolic propolis extract (PE), have been shown to be a safe and effective therapeutic alternative for the treatment of fungal skin and nail diseases. However, the presence of the resin and the physicochemical characteristics of the extract sometimes difficult the reading and determination of breakpoints of the in vitrotests, evidencing the need for alternatives that facilitate the reading. The present study aimed to standardize the use of resazurin in tests of susceptibility of PE with planktonic yeast cells and biofilm forms. The antifungal activity of PE was determined by minimum inhibitory concentration (MIC) and we observed that, for all Candida spp. tested, the most reproducible MIC results were obtained when resazurin was placed after 24 hours of incubation and remained more 24 hourswith yeasts plus PE. For encapsulated yeasts, there was no dye reduction and color transition. Resazurin was also used for the evaluation of minimal biofilm inhibitory concentration and minimal biofilm eradication concentration and it was metabolized and reproducedthe action of PE on Candida biofilms. In addition, microdilution checkerboard plates were made with the dye, which assisted reading the result of the interaction between PE and nystatin. We observed that the resin, the color and the turbidity of the PE slightly changed the color of the resazurin in high concentrations of the extract and did not impair the reading. Therefore, the resazurin standardization tests were proven to be efficient and grounds that it should be used as an auxiliary methodology for reading and interpretation of the susceptibility tests for non-encapsulated yeasts with natural products, which form turbidity or precipitation, such as propolis.

Effect of Silicon dioxide coating of acrylic resin surfaces on Candida albicans adhesion.

Acrylic resin has been used in the manufacture of prostheses, however, in the oral cavity, this material starts to retain microorganisms capable of causing gingival inflammation due its porosities. The aim of this study was to evaluate the influence of the use of silicon dioxide as a coating layer applied onto acrylic resin, on the adhesion of Candida albicans (Ca). After the incubation period in Sabouraud Dextrose Broth, a total of 1 ml of the Ca suspension was added to plate wells, each well containing a specimen of acrylic resin. The adhesion ability of Ca on acrylic resin was determined by counting colonies. Three groups (n = 6) of acrylic resin were assessed: with polishing (RP); without polishing (RW); with polishing and coating layer of silicon dioxide (RPC). Ca deposited on the surface of the acrylic resin was also observed using Scanning Electron Microscopy (SEM). Statistical assessment by Kruskal-Wallis and Student-Newman-Keuls Method were done (α = 2%). There was significant difference among the groups. The RPC group showed the lowest growth, with an average of 5.59 Log CFU/cm 2 ; there was a statistically significant difference in relation to group RW, which presented a growth of 6.07 Log CFU/cm 2 and to group RP with 5.91 Log CFU/cm 2 (p < 000.1). SEM images demonstrated that in the RP and RPC group, the surface of the resin had greater regularity, and smaller number of microorganisms. The application of silicon dioxide coating on acrylic resin appears to be a promising alternative, and its use can help in reducing the adhesion of Ca in prostheses.

Microbiological and virulence aspects of Rhodotorula mucilaginosa.

We aimed to characterize microbiologically clinical isolates of R. mucilaginosa isolated from colonization of a patient with chronic renal disease (CKD), as well as to evaluate their phylogeny, antifungal susceptibility, virulence, and pathogenicity in order to infer the potential to become a possible infective agent. For this study, two isolates of R. mucilaginosa from oral colonization of a CKD patient were isolated, identified and characterized by classical (genotypic and phenotypic) methods. Susceptibility to conventional antifungals was evaluated, followed by biofilm production, measured by different techniques (total biomass, metabolic activity, colony forming units and extracellular matrix quantification). Finally, the pathogenicity of yeast was evaluated by infection of Tenebrio molitor larvae. All isolates were resistant to azole and sensitive to polyenes and they were able to adhere and form biofilm on the abiotic surface of polystyrene. In general, similar profiles among isolates were observed over the observed periods (2, 24, 48 and 72 hours). Regarding extracellular matrix components of biofilms at different maturation ages, R. mucilaginosa was able to produce eDNA, eRNA, proteins, and polysaccharides that varied according to time and the strain. The death curve in vivo model showed a large reduction in the survival percentage of the larvae was observed in the first 24 hours, with only 40 % survival at the end of the evaluation. We infer that colonization of chronic renal patients by R. mucilaginosa offers a high risk of serious infection. And also emphasize that the correct identification of yeast is the main means for an efficient treatment.

Fatores de virulência e susceptibilidade a antifúngicos de Cryptococcus Spp / Virulence factors and susceptibility to Cryptococcus Spp. antifungals

Criptococose é uma doença grave que afeta tanto imunocomprometidos quanto imunocompetentes, com isso analisar a virulência é fundamental para novas terapêuticas. Objetivo: Analisar a capacidade de virulência e susceptibilidade aos antifúngicos de Cryptococcus spp. isolados de líquor de pacientes de hospital do norte do Paraná. Métodos: A partir de dois isolados clínicos C. neoformans e C. gattii, realizou-se a confirmação da identificação. Para a virulência, avaliou-se o tamanho da cápsula, capacidade de sobrevivência após exposição a neutrófilos, produção de melanina e urease. No antifungigrama por difusão em disco utilizou-se: anfotericina B, cetoconazol, voriconazol, itraconazol e miconazol. Resultados: C. gattii destaca-se por maior desenvolvimento da cápsula além da melhor capacidade de sobreviver a fagocitose em relação ao C. neoformans. No antifungigrama, ambos os isolados se apresentam sensíveis às drogas estudadas. Conclusão: Esses achados contribuem para a compreensão das diferentes patogêneses entre C. gattii e C. neoformans.

Cryptococcosis is a serious disease that can affect both immunocompromised and immunocompetent individuals, thus the virulence analysis is fundamental for the development of new treatments. Objective: To analyze the virulence and susceptibility of Cryptococcus spp. isolated from cerebrospinal fluid of patients from a hospital in the north of Paraná. Methods: From two clinical isolates, C. neoformans and C. gattii were confirmed and identified. For virulence, capsule size, survival capacity after exposure to neutrophils, melanin production and urease were evaluated. In the disc-diffusion method, the following antifungals were used: amphotericin B, ketoconazole, voriconazole, itraconazole and miconazole Results: It was observed that C. gattii presents greater results for development of the capsule beside presenting the best ability to survive phagocytosis in relation to C. neoformans. In the disc-diffusion method, both isolates presented sensitivity to the studied drugs. Conclusion: These findings contribute to the understanding of the different pathogens between C. gattii and C. neoformans.

Effect of Silicon dioxide coating of acrylic resin surfaces on Candida albicans adhesion

Abstract Acrylic resin has been used in the manufacture of prostheses, however, in the oral cavity, this material starts to retain microorganisms capable of causing gingival inflammation due its porosities. The aim of this study was to evaluate the influence of the use of silicon dioxide as a coating layer applied onto acrylic resin, on the adhesion of Candida albicans (Ca). After the incubation period in Sabouraud Dextrose Broth, a total of 1 ml of the Ca suspension was added to plate wells, each well containing a specimen of acrylic resin. The adhesion ability of Ca on acrylic resin was determined by counting colonies. Three groups (n = 6) of acrylic resin were assessed: with polishing (RP); without polishing (RW); with polishing and coating layer of silicon dioxide (RPC). Ca deposited on the surface of the acrylic resin was also observed using Scanning Electron Microscopy (SEM). Statistical assessment by Kruskal-Wallis and Student-Newman-Keuls Method were done (α = 2%). There was significant difference among the groups. The RPC group showed the lowest growth, with an average of 5.59 Log CFU/cm 2 ; there was a statistically significant difference in relation to group RW, which presented a growth of 6.07 Log CFU/cm 2 and to group RP with 5.91 Log CFU/cm 2 (p < 000.1). SEM images demonstrated that in the RP and RPC group, the surface of the resin had greater regularity, and smaller number of microorganisms. The application of silicon dioxide coating on acrylic resin appears to be a promising alternative, and its use can help in reducing the adhesion of Ca in prostheses.

Implications of the presence of yeasts in tracheobronchial secretions of critically ill intubated patients.

The presence of some microorganisms in the respiratory tract is a known risk factor for the infection of air passages; however, it is not clear whether this holds true for Candida spp. Thus, our objective was to determine the frequency of yeast colonization in the tracheobronchial secretions of critically ill intubated patients and to assess the presence of these yeasts in the infra-cuff region of the endotracheal tube (ET). Patients aged 18 years or older who had been using an endotracheal tube for 48 hours were recruited. Tracheal secretions were collected; after extubation, the ETs were cut into two fragments in the infra-cuff region. One of these fragments was placed in a solution containing antibiotics and sent to the lab for culture and identification of yeasts. The remaining fragment was fixed and subjected to scanning electron microscopy (SEM). In total, 20 patients with an average age of 73.3 years (± 13.1) participated in this study. These patients remained under endotracheal intubation and invasive mechanical ventilation for an average of 6.4 (± 1.8) and 13.5 days (± 15), respectively. Of these patients, 45 % showed respiratory tract colonization by yeasts of the Candida genus, with C. albicans being the most frequently isolated species (66.7 %). Moreover, in almost 90 % of these patients, blastoconidia of the same yeast were found in the infra-cuff portion of the ET, as evidenced by SEM, strongly fixed on the ET surface. Yeasts isolated from both the infra-cuff region and the tracheobronchial secretions were susceptible to amphotericin B and fluconazole. In conclusion, our results show that the frequency of colonization by yeasts of the Candida genus in the tracheobronchial secretions of intubated patients within 48 hours is high, and that these species can also be found as a biofilm on the ET surface.

Occurrence of dermatophytoses in patients from the Sistema Único de Saúde.

BACKGROUND: Dermatophytosis is a cutaneous disease caused by filamentous keratinophilic fungi belonging to the genera Trichophyton, Microsporum and Epidermophyton, which present a high prevalence in the general population, being among the most common mycoses affecting about 20% of the world's population. OBJECTIVE: To carry out the epidemiological survey of cases of dermatophytosis in patients from the Sistema Único de Saúde in a regional Laboratory in the period of 5 years (2009 to 2013). METHODS: A retrospective study (January 2009 to December 2013) was carried out with a qualitative and quantitative design, through the registry book of the laboratory, Mycology Sector, where cases of patients with suspected dermatomycosis were analyzed. RESULTS: In a 5-year period, a total of 4467 cases were suspected of having a fungal infection. Of these, 68.74% (3071) cases were of dermatomycosis. In relation to cultures with fungal growth, 12.54% (385 cases) were dermatophyte fungi and 7.97% (245 cases) non-dermatophyte fungi were isolated. Among the species identified, there was a higher prevalence of T. rubrum complex (75%), T. mentagrophytes complex (11.68%) and M. canis (7.01%). Regarding the sites analyzed, nail involvement was the most frequent in 75% of the cases. STUDY LIMITATIONS: This work is representative in the studied region. CONCLUSIONS: Dermatomycosis samples are the most frequent among all samples of fungal infections from these patients, with the nail being the most affected area and the fungi T. rubrum complex and T. mentagrophytes complex the most frequent.

Occurrence of dermatophytoses in patients from the Sistema Único de Saúde

Abstract: Background: Dermatophytosis is a cutaneous disease caused by filamentous keratinophilic fungi belonging to the genera Trichophyton, Microsporum and Epidermophyton, which present a high prevalence in the general population, being among the most common mycoses affecting about 20% of the world's population. Objective: To carry out the epidemiological survey of cases of dermatophytosis in patients from the Sistema Único de Saúde in a regional Laboratory in the period of 5 years (2009 to 2013). Methods: A retrospective study (January 2009 to December 2013) was carried out with a qualitative and quantitative design, through the registry book of the laboratory, Mycology Sector, where cases of patients with suspected dermatomycosis were analyzed. Results: In a 5-year period, a total of 4467 cases were suspected of having a fungal infection. Of these, 68.74% (3071) cases were of dermatomycosis. In relation to cultures with fungal growth, 12.54% (385 cases) were dermatophyte fungi and 7.97% (245 cases) non-dermatophyte fungi were isolated. Among the species identified, there was a higher prevalence of T. rubrum complex (75%), T. mentagrophytes complex (11.68%) and M. canis (7.01%). Regarding the sites analyzed, nail involvement was the most frequent in 75% of the cases. Study Limitations: This work is representative in the studied region. Conclusions: Dermatomycosis samples are the most frequent among all samples of fungal infections from these patients, with the nail being the most affected area and the fungi T. rubrum complex and T. mentagrophytes complex the most frequent.

Effects of intratracheal Fusarium solani inoculation in immunocompetent mice.

Fusariosis is an infection that is caused by fungi of the Fusarium genus. It is the second most common fungus that is associated with human fungal infections, usually in immunocompromised individuals. The incidence of such infections has been increasing, including in immunocompetent hosts. Studies of host-pathogen interactions are scarce, and the pathophysiology of the disease is unknown. One limitation of such studies is the lack of adequate techniques for mammalian infection, in which no standardized protocols have been established with fungi with a focus on the respiratory tract. The aim of the present study was to assess the first 24 h of infection after the intratracheal inoculation of F. solani microconidia in immunocompetent mice. Colony-forming units (CFU) were counted, and histopathological analysis was performed. Under conditions of high fungal burden, F. solani caused lethal tissue damage in the lungs. Under conditions of low fungal burden, the infection was not lethal, but several alterations of pulmonary tissue and the presence of the fungus in the lungs were observed. No evidence of fungal dissemination was found in the kidneys, spleen, liver, or heart 24 h after infection. The present intratracheal model effectively established fungal infection and appears to be suitable for studies of Fusarium spp.

Candida parapsilosis isolates from burn wounds can penetrate an acellular dermal matrix.

We isolated and identified yeasts from burn wounds and evaluated the ability of Candida parapsilosis isolates from burn wounds to penetrate an acellular dermal matrix (ADM). A prospective study was conducted with patients from the burn treatment center of North Paraná University Hospital in Londrina, Brazil from February 2015 to January 2016. Yeast cultures were obtained from the tissue of burn wounds that had been debrided and cleansed with 2% chlorhexidine. After identification and confirmation of the purity of the culture, the yeasts were placed on ADM fragments and incubated for three or seven days. During the study period, 273 patients were treated, and 36 of these patients fulfilled the inclusion criteria and provided samples for culture. Yeasts were isolated in 19.44% (n = 7) of the cultures, and the following species were identified: C. parapsilosis (57.1%), C. albicans (28.6%), and C. glabrata (14.3%). C. parapsilosis, the most frequent species, was chosen for the ADM tests. We demonstrated active penetration of the ADM by the yeast isolates from burn wounds. C. parapsilosis grew on ADM and penetrated the matrix, indicating that this yeast, which is common in skin and cutaneous wounds, has the potential to colonize and pass through ADM, a medical device that is frequently used to dress and regenerate burn wounds.

Yeasts from skin colonization are able to cross the acellular dermal matrix.

In recent decades, the prognosis for burn patients has improved considerably with the development of specialized care. The acellular dermal matrix (ADM) is a totally artificial acellular device that functions to control water loss, prevent penetration by bacteria and allow migration of endothelial cells and fibroblasts from patient tissues. However, little is known about its effectiveness against yeasts. The present study evaluated the capacity of colonization and migration of some human commensal yeasts. Three clinical isolates from skin scales, identified as Candida parapsilosis, Candida glabrata and Rhodotorula mucilaginosa, were used. Their ability to cross the ADM was evaluated. After three days, all isolates had crossed the ADM. C. parapsilosis showed the lowest growth, while R. mucilaginosa showed intermediate and C. glabrata the highest growth. In the plates incubated for seven days, the growth of C. parapsilosis and C. glabrata increased by 1 log over the third day. All isolates have the capacity to colonize and migrate through the matrix, increasing the potential risk to burn patients, who can develop severe and even fatal infections by invasive fungi.