Ascending renal infection following experimental candiduria by Candida tropicalis in immunocompromised mice.

The present study evaluated renal infection resulting from the implantation of C. tropicalis in the bladder of immunosuppressed mice. Yeasts were implanted in two manners: planktonic and via preformed biofilm on a small catheter fragment (SCF). Renal histopathology and cultures was performed 72 and 144 h after cystotomy was carried out in mice from three groups: group I contained non-contaminated mice implanted with a sterile SCF; group II mice received a sterile SCF plus a yeast suspension containing 1 × 107 yeasts/mL in a planktonic form; group III mice were implanted with a SCF containing preformed C. tropicalis biofilm. Viable yeasts were found in the kidneys of mice from both groups II and III. However, after 72 h the planktonic cells (group II) invaded more quickly than the sessile cells (group III). Over a longer period (144 h), group III exhibited a more invasive infection (50% of the animals presented renal infection and the renal fungal load was 3.2 log10 CFU/g tissue) than in group II, where yeasts were not found. C. tropicalis introduced into the bladder in two ways (in planktonic or biofilm form) were able to reach the kidney and establish a renal fungal infection, causing interstitial disorders. The data of the present study therefore support the hypothesis of an ascending pathway for renal infections by C. tropicalis. Furthermore, the biofilm resulted in a greater and progressive risk of renal infection, attributed to the slow detachment of the yeasts.

Serial systemic candidiasis alters Candida albicans macromorphology associated with enhancement of virulence attributes.

Candida albicans is one of the major pathogens found in superficial and invasive infections. This fungus expresses several virulence factors and fitness attributes that are essential to the pathogenesis. In our previous study using a murine model of serial systemic candidiasis, virulence of the recovered C. albicans was enhanced and several virulence factors were also modified after five successive passages through mice (P1-P5). In this study, we aimed to correlate the different fungal morphologies, as well as the filamentation, invasion, and stress resistance abilities, of the cells recovered after passing through this model of infection with our previous findings regarding virulence. We obtained two colony morphology types from the recovered cells, differing in their peripheral filamentation. The morphotype 1, which presented zero to five filaments in the colony edge, was higher in P2, while morphotype 2, which presented more than five filaments in the colony edge, was predominant from P3 to P5. In general, morphotype 1 showed similar levels regarding filamentation in serum, invasion of agar and cells, and resistance to osmotic, oxidative, and thermal stress in all passages analyzed. The morphotype 2, however, exhibited an enhancement in these abilities over the passages. We observed an accordance with the increased virulence over the passages obtained in our previous study and the increased adaptability profile of morphotype 2. Therefore, we suggest that the behavior observed previously in the pathogenesis and virulence could be attributed, at least in part, to the greater presence and ability of morphotype 2.

The Success of Topical Treatment of Onychomycosis Seems to Be Influenced by Fungal Features.

AIM: To evaluate the topical treatment of onychomycosis using a 10% hydroalcoholic propolis extract (PE) in two aleatorily chosen patients and analyze possible risk factors from hosts including some particularities of the isolated fungi that may justify the outcomes achieved. MATERIALS AND METHODS: A topical treatment, with PE, was started in two cases of toe onychomycosis due to T. rubrum. The in vitro PE antifungal activity against these isolates was confirmed. Moreover, the ability of the fungi to infect the human nail was evaluated also in an ex vivo study, analyzed by histopathology. RESULTS: Within four months, both patients showed evident improvement, but with different outcomes. The possible host-related risk factors justifying the poorer outcome in patient 1 include a longer duration time of onychomycosis (50 years). Some particularities in the T. rubrum strain isolated from this patient in relation to that found in patient 2 were observed: (1) the hypha morphology suggesting a major adaptation of the fungus to the host; (2) a 16 times greater propolis concentration was required in vitro; and (3) a faster ability to start a growth using the nail as the only nutritional source. Additionally, this isolate was more efficient in producing a biofilm on the nail surface. CONCLUSIONS: A partial clinical and complete mycological cure for the two patients was achieved after four months of PE daily use. Despite a complete recovery, a different outcome was observed between both cases. A more persistent onychomycosis, added to greater fungal potential to produce biofilm on the nail, seems to influence greatly the success of a topical treatment with PE.

In vitro interaction of Candida tropicalis biofilm formed on catheter with human cells.

Candida tropicalis has emerged as one of the major Candida non-C. albicans species, in terms of epidemiology and virulence. Despite its virulence, C. tropicalis pathogenic mechanism has yet not been fully defined. The current study aimed to demonstrate the interaction of mature C. tropicalis ATCC 750 biofilm formed on catheter with different human cell lines. In vitro mature (72 h) C. tropicalis biofilms were produced on small catheter fragments (SCF) and were mainly composed by blastoconidia. Then, migration of yeast cells from mature biofilm to human cell surfaces (HeLa and HUVEC) was investigated. After contact with both cell lines, the surface of SCF, containing mature C. tropicalis biofilm, exhibited predominantly the filamentous form. Meanwhile, fresh biofilm formed on human cell surfaces also revealed mainly of blastoconidia involved by extracellular matrix. Total biomass and metabolic activity from the remaining biofilm on SCF surface, after direct contact with human cells, exhibited a significant reduction. Mature C. tropicalis biofilm modified its extracellular matrix components, after contact with human cells. Thus, we described for the first time an easy and simple in vitro model with catheter, which could be a powerful tool for future studies that desires to elucidate the mechanisms involved in C. tropicalis biofilm.

Propolis increases neutrophils response against Candida albicans through the increase of reactive oxygen species.

AIM: To investigate whether Brazilian green propolis improves the immune response against recurrent form isolate recurrent vulvovaginal candidiasis (RVVC) caused by Candida albicans by increasing neutrophil oxidative burst. MATERIALS & METHODS: We evaluated oxidant species production, oxygen consumption, microbicidal activity and myeloperoxidase activity in neutrophils previously treated with propolis and activated with different isolates of C. albicans (RVVC), vulvovaginal candidiasis, asymptomatic isolates and the reference strain. RESULTS: Propolis significantly increased oxidant species production, oxygen consumption, microbicidal activity and myeloperoxidase activity of neutrophils against different isolates of C. albicans including RVVC isolate that are considered resistant to the microbicidal activity of neutrophils. CONCLUSION: Brazilian green propolis may increase neutrophil burst oxidative response to RVVC leading to an efficient removal of C. albicans.

Cinnamaldehyde induces changes in the protein profile of Salmonella Typhimurium biofilm.

The effect of cinnamaldehyde against biofilm cells of Salmonella Typhimurium ATCC 14028 was evaluated. We also assessed differential protein patterns that were expressed by biofilms compared with planktonic cells and protein expression by cinnamaldehyde-treated biofilms cells. This compound decreased biofilm biomass and metabolic activity of biofilms at both concentrations tested. Cinnamaldehyde treatment reduced the number of attached cells in polypropylene, reflected by colony count and scanning electron microscopy. The proteomic analysis of biofilms compared with planktonic cells indicated that several proteins were upregulated or downregulated, especially proteins that are involved in energy metabolism. Peroxiredoxin, ATP synthase alpha chain protein, conjugal transfer nickase/helicase TraI and elongation factor G were upregulated in untreated-biofilm cells, and their expression decreased as a function of cinnamaldehyde treatment. Cinnamaldehyde had antibiofilm activity, and several differentially expressed proteins identified provide potential and interesting targets to explore new control strategies for S. Typhimurium biofilms.

Antibiofilm activity of propolis extract on Fusarium species from onychomycosis.

AIM: The present study evaluated the capacity of three species of Fusarium isolated from onychomycosis to form biofilms and the antibiofilm effect of propolis extract on these biofilms. MATERIALS & METHODS: The biofilms and antibiofilm effects were evaluated by quantifying the colony-forming units, mitochondrial metabolic activity assays, total biomass by crystal violet staining and scanning electron microscopy. RESULTS: Propolis extract demonstrated significant antibiofilm efficiency on Fusarium spp. isolates and reduced F. solani, F. oxysporum and F. subglutinans mature biofilms. CONCLUSION: Propolis extract can be an alternative topical treatment of onychomycosis caused by Fusarium spp.

Antifungal activities of diphenyl diselenide and ebselen against echinocandin-susceptible and -resistant strains of Candida parapsilosis.

We evaluated the in vitro antifungal activity of diphenyl diselenide and ebselen against echinocandin-susceptible and -resistant strains of Candida parapsilosis using the broth microdilution method. Diphenyl diselenide (MIC range =1-8 µg/mL) and ebselen (MIC range =0.25-4 µg/mL) showed in vitro activity against echinocandin-susceptible isolates. However, ebselen also showed the highest antifungal activity against echinocandin-resistant strains (MIC range =0.06-4 µg/mL). This study demonstrated that the antifungal potential of diphenyl diselenide and ebselen deserves further investigation using in vivo experimental protocols.

Microbicidal activity of neutrophils is inhibited by isolates from recurrent vaginal candidiasis (RVVC) caused by Candida albicans through fungal thioredoxin reductase.

Vulvovaginal candidiasis (VVC) is characterized by an infection of the vulva and vagina, mainly caused by Candida albicans, a commensal microorganism that inhabits the vaginal, digestive, and respiratory mucosae. Vulvovaginal candidiasis affects approximately 75% of women, and 5% develop the recurrent form (RVVC). The aim of the present study was to evaluate whether neutrophils microbicidal response is triggered when activated with RVVC isolates caused by C. albicans. Our results showed that RVVC isolates induced neutrophil migration but significantly decrease the microbicidal activity of neutrophils, compared with VVC and ASS isolates. The microbicidal activity of neutrophils is highly dependent on the production of reactive oxygen species/reactive nitrogen species (ROS/RNS). However, this isolate induced detoxification of ROS/RNS produced by neutrophils, reflected by the high level of thiol groups and by the oxygen consumption. Therefore, RVVC isolates induced biochemical changes in the inflammatory response triggered by neutrophils, and these effects were mainly related to the detoxification of ROS/RNS through the thioredoxin reductase (TR), a key antioxidant enzyme in fungi. This might be one of the resistance mechanisms triggered by RVVC caused by C. albicans.

Microparticles containing propolis and metronidazole: in vitro characterization, release study and antimicrobial activity against periodontal pathogens.

Ethylcellulose microparticles containing metronidazole and propolis extractive solution were prepared and evaluated in vitro against periodontal pathogens. Scanning electron microscopy, particle size analysis, drug entrapment efficiency and drug release of microparticles were determined. The antimicrobial activity of microparticles was evaluated against microorganisms of periodontal importance (Enterococcus faecalis, Streptococcus pyogenes, Streptococcus mutans, Staphylococcus aureus, Klebsiella pneumoniae and Escherichia coli). It was obtained particles with regular morphology, mean diameter of 1.23 µm, and entrapment efficiency for propolis and metronidazole were 91.41% and 22.23%, respectively. In vitro release studies of propolis and metronidazole from microparticles showed prolonged drug release and controlled by Fickian diffusion. Both propolis and metronidazole displayed activity against the tested strains. Moreover, the results showed that the strains of E. faecalis, S. pyogenes and S. mutans were more susceptible to the propolis and E. faecalis to the metronidazole. It was also observed that the amount of metronidazole to inhibit the microorganism strains in the physical mixture with propolis was smaller than in the metronidazole alone, suggesting potentiation effect between propolis and metronidazole. These microparticles would be useful for developing intermediary or eventual dosage form to be administered into the periodontal pocket more easily and safely.

Susceptibilidad de levaduras vaginales a los antifúngicos más utilizados en Maringá, Paraná, Brasil / Susceptibility to vaginal yeast in most used antifungal in Maringá, Paraná, Brazil

Se determinó la susceptibilidad antifúngica in vitro de 78 cepas de levaduras aisladas de mujeres de la ciudad de Maringá/Paraná, Brasil, con candidiasis vulvovaginal (CVV), atendidas en el Laboratorio de Enseñanza e Investigación en Análisis Clínicos (LEPAC) de la Universidad Estatal de Maringá, desde el 1 de enero 2005 al 31 de diciembre 2006. Su sensibilidad in vitro fue investigada por el método de microdilución frente a ketoconazol (KETO), fluconazol (FLU), itraconazol (ITRA), nistatina (NIS) y anfotericina B (AMB). Para KETO, 41,5% de las cepas de C. albicans y 96% de Candida no-albicans presentaron resistencia (100% de C. glabrata) y para FLU solamente el 3,8% de los aislamientos de C. albicans y el 8,0% de C. glabrata fueron resistentes. Sólo 1,9% de las cepas de C. albicans y 20% de las de C. no-albicans fueron resistentes a ITRA y el 5,7% de las C. albicans y el 8% de las C. no-albicans (sólo C. glabrata) fueron resistentes a AMB. No hubo aislamientos resistentes a NIST, pero sí una elevada frecuencia de sensibilidad dosis dependiente "in vitro". Estos datos avalan la creciente necesidad de la realización de pruebas de identificación y susceptibilidad in vitro a los antifúngicos para establecer el correcto tratamiento de la CVV.

In vitro antifungal susceptibility was determined in the 78 yeasts isolated from patients with vulvovaginal candidiasis (VVC) from the city of Maringá/Parana/Brazil, assisted in the Laboratory of Teaching and Research in Clinical Analysis of the State University of Maringá, from 01 January 2005 to December 31, 2006. Its sensibility in vitro was tested according to microdilution method in front of ketoconazol (KETO), fluconazole (FLU), itraconazole (ITRA), nistatin (NIS) and amphotericin B (AMB). For KET, 41.5% of the C. albicans and 96.0% of the C. non-albicans showed resistance (100.0% of C. glabrata) and for FLU, only 3.8% of the isolates of C. albicans and 8.0% of C. glabrata showed resistance. Only 1.9% of the C. albicans and 20% of the C. no-albicans were resistant. For AMB, 5.7% of the C. albicans and 8% of the C. no-albicans (only C. glabrata), were resistant. There were no isolations resistant from NIST, however, there was a high frequency of dose-dependent sensibility (SDD) in vitro. These data makes it possible to confirm the growing necessity of the performance of identification tests and in vitro antifungal susceptibility to antifungals to establish the correct treatment of CVV.

Characteristics of biofilm formation by Candida tropicalis and antifungal resistance.

Candida tropicalis is a common species related to nosocomial candidemia and candiduria. Most Candida spp. infections are associated with biofilm formation on implanted medical devices or on host epithelial cell surfaces. Sessile cells display phenotypic traits dramatically different from those of their free-living, planktonic counterparts, such as increased resistance to antimicrobial agents and to host defenses. The characteristics of C. tropicalis biofilm formation in vitro are described. By an XTT-reduction assay, an increase in metabolic activity was observed up to 24 h of biofilm formation, and this activity showed a linear relationship with sessile cell density. Scanning electron microscopy was used to further characterize C. tropicalis biofilms. The initial adherence of yeast cells was followed by germination, microcolony formation, filamentation and maturation at 24-48 h. Mature biofilms consisted of a dense network of yeast cells and filamentous forms of C. tropicalis. Increased resistance of sessile cells against fluconazole and amphotericin B was also demonstrated. Real-time reverse transcription-PCR quantification showed that sessile cells overexpressed ERG11 (coding for lanosterol 14 alpha-demethylase) and MDR1 (coding for an efflux protein belonging to the major facilitator superfamily). These mechanisms may contribute to the fluconazole resistance of the C. tropicalis biofilm.

New feather-degrading filamentous fungi.

Among 106 filamentous fungi isolated from poultry farm waste, 13 species belonging to seven genera (Aspergillus, Acremonium, Alternaria, Beauvaria, Curvularia, Paecilomyces, and Penicillium) were able to grow and produce keratinase in stationary cultures using poultry feather powder as the only substrate. The four most efficient keratinase producers were selected for a comparative study of keratinase production in submerged and stationary conditions. The highest keratinolytic activities were produced after 4-6 days of cultivation in submerged conditions: 53.8 +/- 6.1 U/mL (Alternaria tenuissima), 51.2 +/- 5.4 U/mL (Acremonium hyalinulum), 55.4 +/- 5.2 U/mL (Curvularia brachyspora), and 62.8 +/- 4.8 U/mL (Beauveria bassiana). These novel nondermatophytic keratinolytic fungi have potential use in biotechnological processes involving keratin hydrolysis. The results of this work contribute to show that keratinolytic activity is relatively widespread among common filamentous fungi and may have an important rule in feather decomposition in natural settings.

A simplified technique for evaluating the adherence of yeasts to human vaginal epithelial cells.

Thousands of women all over the world annually suffer of vulvovaginal candidiasis (VVC), an infection caused by yeasts, which mainly affect the mucosa of the vulva and vagina. The adherence of the yeasts to the mucosae is an essential step for colonization and predisposes the start of the infectious process. In this study, a technique capable of evaluating the adhesion of yeasts to human vaginal epithelial cells (HVEC) was employed. Twenty-five vaginal yeast isolates (10 Candida albicans, nine C. glabrata, two C. parapsilosis, one C. tropicalis, two Saccharomyces cerevisiae, and one Trichosporon sp.) were evaluated. A suspension of each yeast was co-incubated with HVEC obtained from a healthy donor in the ovulatory phase. After 1 hr, smears were made, stained with crystal violet and Papanicolaou, and the number of yeasts that adhered to 600 HVEC was evaluated. The adhesion of C. albicans was significantly greater than that of the other species and occurred mainly in the intermediate HVEC, rather than the superficial. In addition, the proposed technique, easy to execute and of low cost showed to be reproducible and enables the determination of the adherence capacity of different isolates, whose adhesion was confirmed by scanning electron microscopy.

Correlation of Candida species and symptoms among patients with vulvovaginal candidiasis in Maringá, Paraná, Brazil.

Vulvovaginal candidiasis (VVC) is an infection caused by abnormal yeast growth in the mucosa of the female genital tract which is commonly diagnosed in gynecology. The aim of this study was to correlate the frequency of yeasts and their respective species in asymptomatic women with different clinical manifestation of VVC; evaluate possible relationships between number of fungus colonies and symptoms in this pathology. All patients who visited the laboratory within a period of five months, for routine examinations of vaginal secretion, independent of the presence or absence of symptoms of VVC were included in this study. Of these, women with immunodeficiency or with an infection of the genital tract by another agent were excluded. Candida albicans was the most frequently yeast isolated (60%). Among non-C. albicans yeasts, 61.5% were isolated of the asymptomatic women, 38.7% from patients with VVC and 11.1% of those from patients with RVVC. C. albicans was associated with symptoms of VVC and while, the presence of non-C. albicans yeasts with asymptomatic women. However, there was no association between the number of fungal colonies and symptoms.