Altered phagocytosis and morphogenesis of phenotypic switching-derived strains of the pathogenic Candida tropicalis co-cultured with phagocytic cells.

BACKGROUND AND OBJECTIVE: Candida tropicalis is among the most prevalent human pathogenic yeast species. Switch states of C. tropicalis differ in virulence traits. Here, we evaluate the effect of phenotypic switching on phagocytosis and yeast-hyphae transition in C. tropicalis. METHODS: C. tropicalis morphotypes included a clinical strain and two switch strains (rough variant and rough revertant). In vitro, phagocytosis assay was performed using peritoneal macrophages and hemocytes. The proportion of hyphal cells was ascertained by scoring morphology using optical microscopy. Expression of the WOR1 (White-opaque regulator 1) and EFG1 (Enhanced filamentous growth protein 1) was determined by quantitative PCR. RESULTS: The rough variant was more resistant to in vitro phagocytosis by peritoneal macrophages than that observed for the clinical strain, while hemocytes phagocytosed clinical and rough variant to the same extent. The rough revertant was more phagocytosed than the clinical strain by both phagocytes. During co-incubation with phagocytic cells, the clinical strain of C. tropicalis exists mainly as blastoconidia. The co-culture of the rough variant with macrophages resulted in a higher percentage of hyphae than blastoconidia cells, while in co-culture with hemocytes, no differences were observed between the percentage of hyphae and blastoconidia. The expression levels of WOR1 in the rough variant co-cultured with phagocytes were significantly higher than they were in the clinical strain. CONCLUSIONS: Differences on phagocytosis and hyphal growth between switch states cells of C. tropicalis co-cultured with phagocytic cells were observed. The pronounced hyphal growth may affect the complex host-pathogen relationship and favor the pathogen to escape phagocytosis. The pleiotropic effects of phenotypic switching suggest that this event may contribute to the success of infection associated with C. tropicalis.

Adhesion and biofilm formation by the opportunistic pathogen Candida tropicalis: what do we know?

Candida tropicalis is among the most important Candida species in terms of epidemiology, virulence and resistance. Considering the increase in C. tropicalis incidence and high rates of mortality associated with this species, knowledge of its adhesion and biofilm formation abilities is needed. These traits determine the persistence and survival of yeast on different indwelling medical devices and host sites. C. tropicalis is among the most adherent Candida species, and it has been described as a strong biofilm producer. Environmental factors, phenotypic switching and quorum sensing molecules can affect adhesion and biofilm growth. C. tropicalis can form sexual biofilms, which are promoted by mating pheromones. C. tropicalis biofilms are regulated by a wide and complex network of genes and signaling pathways that are currently poorly understood. Morphological studies showed improved biofilm architecture, which was related to the expression of several hypha-specific genes. Based on recent updates, research is still needed to increase our knowledge on the genetic network of adhesion and biofilm formation by C. tropicalis, as well as the protein diversity that mediates interactions with inert materials and biological surfaces. Here, we have reviewed the main aspects related to adhesion and biofilm formation in C. tropicalis and summarized current knowledge on the significance of these virulence factors in this opportunistic species.

Antibacterial efficacy of Enterococcus microencapsulated bacteriocin on Listeria monocytogenes, Listeria innocua and Listeria ivanovi.

This study focused on the microencapsulation of enterocin from Enterococcus durans (E. durans MF5) in whey powder (WP) using a spray-drying technique followed by the evaluation of how complexation can preserve the enterocin structure and antimicrobial activity against food-borne pathogens. Crude enterocin samples (1 and 5%) were microencapsulated in 10% WP. The antimicrobial activity of unencapsulated (crude) enterocin and microencapsulated enterocin was tested against the target bacteria Salmonella Typhimurium, Escherichia coli, Listeria monocytogenes, Listeria innocua, and Listeria ivanovi. The microencapsulation yields were 31.66% and 34.16% for concentrations of 1 and 5% enterocin, respectively. There was no significant difference between these concentrations. Microencapsulated enterocin was efficient for up to 12 h of cocultivation with Listeria sp., and the concentration required to inhibit the growth of target bacteria presented values of 6400 AU/mL (arbitrary unit). Microencapsulated enterocin demonstrated enhanced efficacy against Listeria species and E. coli when compared with crude enterocin (p < 0.05). Fourier transform-infrared spectroscopy and differential scanning calorimetry results confirmed the presence of enterocin in the microparticles. Scanning electron microscopy showed cell damage of the target bacteria. The results showed that complexation with WP preserved enterocin antimicrobial activity during spray-drying, indicating its potential use as a food preservative.

Deciphering Colonies of Phenotypic Switching-Derived Morphotypes of the Pathogenic Yeast Candida tropicalis.

BACKGROUND: Phenotypic switching generates fungal colonies with altered morphology and allows pathogens to adapt to changing environments. OBJECTIVE: This study investigated the structure and genetic factors of switched morphotypes colonies in Candida tropicalis. METHODS: Morphotypes of C. tropicalis comprised the clinical strain 49.07 that exhibited smooth colony phenotype and switched (crepe and rough) morphotypes that showed colonies with marked structural variations, including wrinkled surface, depressions areas, and irregular edges (structured morphology). The morphotypes were analyzed for the presence and distribution of the extracellular matrix (ECM) at the ultrastructural level-SEM. The composition of the ECM and the percentage of hyphae in colonies were evaluated. The expression of EFG1 (Enhanced filamentous growth protein 1), WOR1 (White-opaque regulator 1), and BCR1 (Biofilm and cell wall regulator 1) in the morphotypes was measured by RT-qPCR. RESULTS: Colonies of the switched variants exhibited distinct arrangements of ECM compared to the smooth phenotype (clinical strain). In addition, rough variant colonies showed higher amounts of total carbohydrates and proteins in ECM (p < 0.05). Switched (crepe and rough) colonies exhibited a higher percentage of hyphae throughout their development (p < 0.05). The mRNA expression levels of EFG1, WOR1, and BCR1 in the rough morphotype were significantly higher than they were in the smooth morphotype. In addition, there was a positive correlation between the expression of these genes and filamentation (hyphae formation) of the rough morphotype (r2 > 0.9472, p < 0.05). CONCLUSION: Structural variations in switched morphotypes colonies of C. tropicalis seem to be associated with increased hyphae growth and the amount and distribution of ECM. Switched colonies have distinct expressions of the EFG1, WOR1, and BCR1 master regulators genes.

Changes in Adhesion of Candida tropicalis Clinical Isolates Exhibiting Switch Phenotypes to Polystyrene and HeLa Cells.

BACKGROUND: Candida tropicalis is an important human pathogen that can undergo multiple forms of phenotypic switching. AIM: We aimed to evaluate the effect of phenotypic switching on the adhesion ability of C. tropicalis. METHODS: C. tropicalis morphotypes included parental phenotypes (clinical isolates) and switch phenotypes (crepe, revertant of crepe-CR, rough, revertant of rough-RR, irregular center and revertant of irregular center-ICR). Adhesion to polystyrene and HeLa cells was determined by crystal violet assay. The percentage of HeLa cells with adhered yeasts and the number of adhered yeasts per HeLa cell were determined by light microscopy. Filamentation among adhered cells was assessed by direct counting. RESULTS: On polystyrene, 60% of the switch strains showed difference (p < 0.05) on adhesion ability compared to their parental counterpart strains, and altered thickness of adhered cells layers. Filamentation was increased among adhered cells of the switched strains compared to parental strains. A positive correlation was observed between adhesion on polystyrene and filamentation for morphotypes of the system 49.07. The majority of the switched strains showed higher adhesion capability to HeLa cells in comparison to the adherence of the clinical strains. All revertant strains showed a higher number of yeast cells per HeLa cell compared to their variant counterparts (p < 0.05), with exception of the ICR. CONCLUSIONS: Our findings indicate that switching events in C. tropicalis affect adhesion and filamentation of adhered cells on polystyrene and HeLa cells. The rise of switch strains with increased adhesion ability may contribute to the success of infection associated with C. tropicalis.

Effect of phenotypic switching on biofilm traits in Candida tropicalis.

Candida tropicalis can undergo multiple forms of phenotypic switching. We have reported a switching system in C. tropicalis that is associated with changes in virulence attributes. We aimed to assess biofilm formation by distinct switch states of C. tropicalis and evaluate whether their sessile cells exhibit altered virulence traits. C. tropicalis strains included the parental phenotype (a clinical isolate) and four switch phenotypes (crepe, rough, revertant of crepe and revertant of rough). Biofilm formation and adhesion capability of sessile cells on polystyrene were assessed through quantification of total biomass. Filamentous forms were characterized by direct counting of sessile cells. A virulence assay was conducted using the Galleria mellonella infection model. Switch variants (crepe and rough) and their revertant counterparts produced higher biofilm biomass (P < 0.05) than the parental strain. Additionally, filamentous forms were enriched among sessile cells of switched strains compared to those observed for sessile cells of the parental strain, with the exception of the revertant of rough. Sessile cells of switched strains showed higher adhesion to polystyrene compared to the parental strain. Sessile cells of the crepe variant and its revertant strain (RC) exhibited higher virulence against G. mellonella larvae than sessile cells of the parental strain. Our findings indicate that switching events in C. tropicalis affect biofilm development and that sessile cells of distinct switch states may exhibit increased adhesion ability and enhanced virulence towards G. mellonella larvae.

Antimicrobial activity of enterocins against Listeria sp. and other food spoilage bacteria.

OBJECTIVE: To determine bacteriocin producers and the prevalence of structural enterocin genes and to detect the spectrum of activity against foodborne pathogens, from isolates of Enterococcus faecium and Enterococcus faecalis that were isolated from food and the environment. RESULTS: The entA, entB, entP, ent1071 and entX genes, which encode enterocins were the most frequently observed. Enterocins were thermostable, proteinaceous, and resistant to catalase. None of the isolates produced hemolysin, and inhibition resulting from bacteriophage lysis was excluded. The bactericidal effect of enterocins against L. innocua 12612 was determined by optical density and colony forming units. For the activity spectrum, elimination of mainly Listeria strains, Bacillus sp. and clinical enterococci, was observed. Imaging with scanning electron microscopy after treatment with enterocin Efm22 showed irregular rod-shaped cells and loss of cellular integrity. CONCLUSIONS: The isolates evaluated in this study are candidates for the production of enterocins that will be used as food biopreservatives, because they have high anti-listerial activity even after 24 h of experimentation, and used in the pharmaceutical area because they inhibit clinical microorganisms.

Assessment of vancomycin resistance transfer among enterococci of clinical importance in milk matrix.

Dissemination of vancomycin resistance in enterococci has been associated with horizontal transfer of mobile genetic elements. Aim of the study was to evaluate if milk matrix is a suitable environment to support transferability of vancomycin resistance (vanA) gene from clinical vancomycin-resistant Enterococcus faecium to vancomycin-sensitive Enterococcus faecalis. Enterococci strains were firstly screened for the presence of cpd (inducible sex pheromone determinant) gene, vanA and tetL genes (vancomycin and tetracycline resistance markers, respectively) and the gelE (extracellular metalloendopeptidase) gene to define the mating pairs. Based on these selection markers, we investigated the transferability of eight plasmid-borne vanA harbored by E. faecium (vanA+, cpd-, tetL- and gelE-) into two E. faecalis (vanA-, cpd+, tetL + and gelE+) recipient strains in milk matrix. The strains were mated in a 1:1 ratio in 7% reconstituted milk and incubated at 37 °C. Transconjugants emerged from all 16 matings within 2 h of incubation and were evidenced by dual antibiotic resistance (vancomycin and tetracycline). The vancomycin-resistance of trasconjugants was maintained even after ten subsequent passages on nonselective medium. Transconjugants were positive for vanA, tetL and gelE genes. This study indicates milk matrix as suitable environment to support gene exchange between Enterococcus species.

Inhibitory effect of bacteriocins from enterococci on developing and preformed biofilms of Listeria monocytogenes, Listeria ivanovii and Listeria innocua.

The biofilm-forming ability of Listeria spp. is a concern to the food industry and health sectors. The aim of this study was to verify the inhibitory activity of bacteriocins produced by enterococci (Enterococcus faecium 20, 22 and 24 and Enterococcus faecalis 27) on developing biofilm and preformed biofilm of Listeria species. Bacteriocins were partially purified from cell free supernatant (CFS). L. monocytogenes 2032, L. innocua 2050 and L. ivanovii 2056 were selected to analyse the inhibitory effect of bacteriocins on biofilm biomass (crystal violet staining) and biofilm viability (XTT-reduction). The biomass of the developing and preformed biofilms of Listeria species were reduced (p < 0.05) in the presence of all bacteriocins tested. Overall, the reduction in biofilm biomass of developing biofilms was up to 87.4% for bacteriocin produced by E. faecium 22 (CFS22) against L. ivanovii and up to 87.1% for CFS22 against L. monocytogenes. These findings are in accordance with those observed in confocal microscopy analysis. Most of the CFS-containing bacteriocin (CFS22, CFS24, CFS27) were effective at decreasing the viability of biofilm cells from all Listeria species. The highest reduction in viability was observed for L. monocytogenes preformed biofilm cells (up to 98.7%), evidenced by fluorescence microscopy of propidium iodide-labelled cells. Scanning electron microscopy showed that cells of biofilm-treated bacteriocins displayed degenerative changes that may be indicative of cellular leakages. This study suggests that bacteriocins produced by enterococci have prospective applications to prevent biofilm formation and/or to reduce cell viability of formed biofilms of distinct Listeria species.

Hemolytic capability and expression of a putative haem oxygenase-encoding gene by blood isolates of Candida tropicalis are influenced by iron deprivation and the presence of hemoglobin and erythrocytes.

Although hemolytic activity is known to be a putative virulence factor contributing to candidal pathogenesis, its production by Candida tropicalis, a species closely related to Candida albicans, is poor understood. The present study was undertaken to evaluate the hemolytic activity and the expression level of a putative haem oxygenase encoding gene by blood isolates of C. tropicalis following growth in iron deprivation, and in the presence of hemoglobin and erythrocytes. The lowest values of hemolytic activity were observed in cell-free culture supernatants of isolates growing in iron-restricted medium (RPMI medium and RPMI medium supplemented with iron chelator bathophenanthrolindisulphonic acid). Hemolysis was increased in the presence of either hemoglobin or erythrocytes. Reverse transcriptase PCR analysis showed that the putative haem oxygenase encoding gene (CtHMX1), potentially related with iron uptake, was up-regulated (p < 0.001) following growth in iron deprivation and in the presence of hemoglobin; CtHMX1 was repressed in the presence of human erythrocytes (p < 0.001). Our data suggest that hemoglobin had positive effect in the production of hemolytic factor and gene expression related to iron uptake in C. tropicalis.

Effects of human blood red cells on the haemolytic capability of clinical isolates of Candida tropicalis.

BACKGROUND: Candida tropicalis is an increasingly important human pathogen associated with high mortality rates; however, little is known regarding the virulence properties of C. tropicalis, particularly the production of haemolytic factor. Although Candida spp may acquire iron from human blood red cells (RBCs) by producing a haemolytic factor that promotes cell lyses, at present there are no data regarding the effect of RBCs on the production of haemolytic molecules. The present study was undertaken to evaluate the role of human red blood cells on the production haemolytic factor by C. tropicalis; in addition, the transcription levels of a putative haemolysin-like protein gene (HLPt) were also analysed. RESULTS: C. tropicalis isolates produced a haemolytic factor following growth in either the absence or presence of RBCs; however, distinct levels of haemolysis were observed, with 60% of the isolates exhibiting a significant increase in the production of haemolytic factor when grown in the presence of human RBCs. All isolates in which the putative HLPt gene was up-regulated in presence of human RBCs, ranging from 1.044 to 6.965-fold, also exhibited higher haemolytic activity following growth in the presence of RBCs compared to that observed in the absence of RBCs. CONCLUSIONS: We propose that human RBCs may induce changes in the phenotypic expression of haemolytic factor and in transcriptional levels of the putative C. tropicalis HLPt gene in an isolate-dependent fashion.

Phenotypic switching in Candida tropicalis: association with modification of putative virulence attributes and antifungal drug sensitivity.

Although Candida tropicalis has become an increasingly important human pathogen, little is known regarding its potential to cause disease. In this study we evaluated the phenotypic switching ability of C. tropicalis and analyzed the effect of switching on biological properties related to virulence factors. We demonstrated that C. tropicalis switched spontaneously, reversibly and at high frequency (10(-1) to 10(-3)) when grown on yeast extract-peptone-D-glucose (YPD) agar medium. Phenotypic switching in five clinical isolates of C. tropicalis resulted in colonies exhibiting the following morphologies: crepe, rough, crater, irregular center, mycelial and diffuse. The majority of the variant colonies were associated with higher percentages of filamentous growth relative to their parental unswitched isolates. Significant differences (P < 0.05) in the production of hemolytic factor were found between most of the switched variants and their respective parental counterparts. Variant colonies exhibiting the crepe (derived from isolates 49.07 and 100.10) and rough phenotype (derived from isolate 49.07) had higher biofilm formation than their parental counterparts exhibiting a smooth dome surface (P < 0.05). Our data revealed that switching was correlated with changes in the in vitro minimum inhibitory concentrations (MICs) of a subset of the switched variants phenotypes to itraconazole. While the MIC to itraconazole was higher for crepe variant compared with its parental isolate 49.07, the rough variant of 100.10 had a lower MIC to this antifungal agent. The presented data support the role of phenotypic switching in promoting changes in phenotypic expression of putative virulence traits and itraconazole susceptibility of clinical isolates of C. tropicalis.

Hemolytic factor production by clinical isolates of Candida species.

Most cases of fungal bloodstream infections (BIs) are attributed to Candida albicans; however, non-Candida albicans Candida species have recently been identified as common pathogens. Although hemolytic factor is known to be putative virulence factor contributing to pathogenicity in Candida species, its production is poorly evaluated. The present study was undertaken to analyze the production of hemolytic factor by C. albicans (10), C. tropicalis (13), and C. parapsilosis (8) isolates associated with BIs. Data of hemolysis zones on plate assay revealed that the majority of C. albicans isolates produced mild hemolytic activity whereas the majority of C. tropicalis produced strong activity. None of the tested C. parapsilosis isolates exhibited hemolysis on plate assay. We also evaluated the hemolytic activity in the cell-free broth. There were no significant differences (P > 0.05) in the secreted hemolytic activity among intra-species isolates. Different levels of secreted hemolytic factor were observed for Candida species, where C. tropicalis exhibited the highest production of hemolytic factor (P < 0.05) followed by C. albicans and C. parapsilosis. Inhibition of hemolysis (up to 89.12 %) from culture supernatant, following incubation with the lectin Concanavalin A (Con A), was observed for all three Candida species. This finding suggests that the secreted hemolytic factor of C. tropicalis and C. parapsilosis may be a mannoprotein, similar to that described for C. albicans.

Nosocomial bloodstream Candida infections in a tertiary-care hospital in South Brazil: a 4-year survey.

The aims of this study were to evaluate the epidemiology of nosocomial candidemia in a tertiary hospital in South Brazil and the in vitro antifungal susceptibility of isolates. Blood strains from 108 patients were identified by PCR-based method. Some 30.5 % of candidemia were caused by Candida tropicalis, 28.7 % were due to Candida albicans, 24.1 % with Candida parapsilosis sensu stricto, 8.3 % with Candida glabrata sensu lato, 1.8 % involved Candida krusei and 6.6 % with other species. Candidemia was more common in intensive care unit settings (66 %). In vitro susceptibility to antifungal drugs was determined by a microdilution method; and new species-specific clinical breakpoints for fluconazole and voriconazole were applied. Overall susceptibility rates were 100 % for itraconazole, 91 % for fluconazole, 98 % for voriconazole and 99 % for amphotericin B. Fluconazole resistance was mostly among C. parapsilosis sensu stricto isolates (26.9 %). Most of the findings reported here agreed with epidemiological features common to other tertiary hospitals in Brazil; but also revealed some peculiarities, such as a high frequency of C. tropicalis associated with candidemia. Besides, high rate of fluconazole resistance among C. parapsilosis stricto sensu isolates was obtained when applying the new species-specific clinical breakpoints.

Phenotypic switching in Candida tropicalis alters host-pathogen interactions in a Galleria mellonella infection model.

Candida tropicalis is a human pathogen associated with high mortality rates. We have reported a switching system in C. tropicalis consisting of five morphotypes - the parental, switch variant (crepe and rough), and revertant (crepe and rough) strains, which exhibited altered virulence in a Galleria mellonella model. Here, we evaluate whether switching events may alter host-pathogen interactions by comparing the attributes of the innate responses to the various states. All switched strains induced higher melanization in G. mellonella larvae than that induced by the parental strain. The galiomicin expression was higher in the larvae infected with the crepe and rough morphotypes than that in the larvae infected with the parental strain. Hemocytes preferentially phagocytosed crepe variant cells over parental cells in vitro. In contrast, the rough variant cells were less phagocytosed than the parental strain. The hemocyte density was decreased in the larvae infected with the crepe variant compared to that in the larvae infected with the parental strain. Interestingly, larvae infected with the revertant of crepe restored the hemocyte density levels that to those observed for larvae infected with the parental strain. Most of the switched strains were more resistant to hemocyte candidacidal activity than the parental strain. These results indicate that the switch states exhibit similarities as well as important differences during infection in a G. mellonella model.

How much do we know about hemolytic capability of pathogenic Candida species?

Hemolytic factor production by pathogenic Candida species is considered an important attribute in promoting survival within the mammal host through the ability to assimilate iron from the hemoglobin-heme group. Hemolytic capability has been evaluated for Candida species based on hemolysis zones on plate assay, analysis of hemolytic activity in liquid culture medium, and hemolysis from cell-free culture broth. The production of hemolytic factor is variable among Candida species, where C. parapsilosis is the less hemolytic species. In general, no intraspecies differences in beta-hemolytic activities are found among isolates belonging to C. albicans, C. glabrata, C. krusei, C. tropicalis, and C. parapsilosis. The production of hemolytic factor by Candida species is affected by several factors such as glucose supplementation in the culture medium, blood source, presence of erythrocytes and hemoglobin, and presence of electrolytes. On the basis of existing achievements, more researches are still needed in order to extend our knowledge about the biochemical nature of hemolytic molecules produced by distinct Candida species, the mechanism of hemolysis, and the molecular basis of the hemolytic factor expression.

Comparison between automated system and PCR-based method for identification and antimicrobial susceptibility profile of clinical Enterococcus spp.

Enterococci are increasingly responsible for nosocomial infections worldwide. This study was undertaken to compare the identification and susceptibility profile using an automated MicrosScan system, PCR-based assay and disk diffusion assay of Enterococcus spp. We evaluated 30 clinical isolates of Enterococcus spp. Isolates were identified by MicrosScan system and PCR-based assay. The detection of antibiotic resistance genes (vancomycin, gentamicin, tetracycline and erythromycin) was also determined by PCR. Antimicrobial susceptibilities to vancomycin (30 µg), gentamicin (120 µg), tetracycline (30 µg) and erythromycin (15 µg) were tested by the automated system and disk diffusion method, and were interpreted according to the criteria recommended in CLSI guidelines. Concerning Enterococcus identification the general agreement between data obtained by the PCR method and by the automatic system was 90.0% (27/30). For all isolates of E. faecium and E. faecalis we observed 100% agreement. Resistance frequencies were higher in E. faecium than E. faecalis. The resistance rates obtained were higher for erythromycin (86.7%), vancomycin (80.0%), tetracycline (43.35) and gentamicin (33.3%). The correlation between disk diffusion and automation revealed an agreement for the majority of the antibiotics with category agreement rates of > 80%. The PCR-based assay, the van(A) gene was detected in 100% of vancomycin resistant enterococci. This assay is simple to conduct and reliable in the identification of clinically relevant enterococci. The data obtained reinforced the need for an improvement of the automated system to identify some enterococci.

In vitro antifungal activity of the flavonoid baicalein against Candida species.

The aim of the present study was to evaluate the in vitro activity of baicalein, the flavone constituent of Scutellaria baicalensis, and synergism of the combination of baicalein and fluconazole against Candida albicans, Candida tropicalis and Candida parapsilosis. The MIC(50) (lowest concentration at which there was 50 % inhibition of growth) of baicalein alone against six Candida strains ranged from 13 to 104 µg ml(-1). For the three species tested, exposure to baicalein at the MIC(50) concentrations obtained for each strain resulted in a high loss of viability. The fluconazole plus baicalein combination markedly reduced the MICs of both drugs for all three strains analysed. In addition, a synergistic effect between baicalein and fluconazole was observed for C. parapsilosis in terms of MIC(50) (fractional inhibitory concentration index = 0.207). Scanning electron microscopy analysis revealed that yeast cells exposed to baicalein at MIC(50) produced a profusely flocculent extracellular material, resembling a biofilm-like structure. In conclusion, these results showed the antifungal capability of baicalein against Candida species and highlight a promising role of baicalein when used in combination with fluconazole against Candida infections.

Ultrastructural architecture of colonies of different morphologies produced by phenotypic switching of a clinical strain of Candida tropicalis and biofilm formation by variant phenotypes.

Candida tropicalis has been identified as one of the most prevalent pathogenic yeast species of the Candida-non-albicans (CNA) group. Study of switching in C. tropicalis has not been the subject of extensive research. Therefore, we investigated switching event and characterized the ultrastructural architecture of different phenotypes and biofilm produced in a C. tropicalis clinical strain. Cells switched heritably, reversibly, and at a high frequency between four phenotypes readily distinguishable by the shape of colonies formed on agar at 25°C. SEM analysis was used to verify the architecture of whole Candida colonies at ultrastructural level. The smooth phenotype (parental phenotype) colony showed a hemispherical shape character, while the semi-smooth was characterized by the presence of shallow marginal depressions. The ring and rough phenotypes exhibited more complex architecture and were characterized by the presence of deep central and peripheral depressions areas. The biofilm-forming ability varied among the switch phenotypes. After 12h incubation, the smooth phenotype formed less biofilm compared to the other phenotypes (P<0.05). The electron microscopy analysis revealed that filamentation (pseudohyphae) was associated with ring and rough colonies. The ultrastructural analysis allowed the observation of the arrangement of individual cells within the colonies. At the deep central and peripheral depressions areas of the ring and rough colonies extracellular material was seen in different arrangements. The data presented here open new avenues to study a possible role for extracellular material in the formation and maintenance of the architecture of switch phenotypes in C. tropicalis. It is therefore essential that more strains be investigated to determine the biological significance of extracellular material in C. tropicalis phenotypic switching phenomenon.