Comparison of the extracellular polymeric substances of Candida albicans and Candida dubliniensis biofilms

Candida albicans and Candida dubliniensis live as benign commensal organisms in the oral cavity of both healthy and unhealthy individuals behaving, under certain conditions, as opportunistic pathogens, causing candidiasis. These two Candida species have been mismatched for years, but recently Candida dubliniensis was recovered from the mouth of imunnosupressed patients and identified as a different species. Candidiasis is usually related with the Candida capacity of forming biofilms on inert or biological surfaces, being this phenotype associated with infections. Biofilms are complex structures of microbial communities attached to a surface, in which microorganisms are embedded in a matrix of extracellular polymeric substances (EPS), composed mainly by proteins and polysaccharides. The biofilm matrix holds the potential of determining possible mechanisms of resistance of Candida biofilms. Several factors are known as affecting the production of EPS, namely, growth medium, growth phase and substratum. This study focused the influence of artificial saliva growth medium in the composition of EPS of biofilms formed by both Candida albicans and Candida dubliniensis strains. Biofilms of one strain of Candida albicans and two strains of Candida dubliniensis were formed in an artificial saliva growth medium (ASGM) and compared with those formed in Sabouraud Dextrose Broth (SDB) and analysed after 48h.The differences between the EPS of biofilms were evaluated (after sonication) in terms of proteins (quantified using the BCA protein assay kit) and polysaccharides (quantified using the phenol-sulphuric method). Proteins were also analysed by SDS-PAGE. In SDB the amount of proteins and polysaccharides in the EPS of biofilms formed by Candida albicans was lower than in the EPS of biofilms formed by Candida dubliniensis strains. In the presence of ASGM the amount of proteins and polysaccharides was similar among the EPS of biofilms of Candida albicans and one of the Candida dubliniensis strains and was lower in biofilms of Candida albicans than in biofilms of the other Candida dubliniensis. Analysis of protein profiles obtained by SDS-PAGE showed that all strains present similar patterns independently of the medium of biofilm formation. Biofilms formed in ASGM originated different amounts of EPS, either in terms of polysaccharides or proteins, compared to the ones formed in SDB. Differences were also found in the profile of extracellular proteins of each strain, depending on the medium

Virulence factor of Candida tropicalis isolated from hospitalized patients

Candida tropicalis has been reported to be one of the Candida species which is most likely to cause bloodstream and urinary tract infections in hospitals. Several virulence factors seem to be responsible for C. tropicalis infections, which present high potential for dissemination and mortality. The aim of this study was to investigate the correlation between different virulence factors (enzymes secretion, adhesion and biofilm formation) and antifungal susceptibility of several C. tropicalis clinical isolates. This study was conducted with 8 isolates of C. tropicalis obtained from urine cultures (4), from blood culture (1) and from central venous catheter (1), from patients admitted to intensive care units at the University Hospital in Maringá, Paraná, Brazil. C. tropicalis ATCC 750 was also used, as a control. Virulence factors evaluated included: adhesion to epithelial cells and silicone, biofilm formation and enzyme production (hemolysins, proteinases, and phospholipases). Susceptibility to fluconazole, itraconazole, voriconazole, and amphotericin B was also determined, by E-test. Regarding adhesion, it can be highlighted that C. tropicalis adhered significantly more (p<0.05) to epithelial cells than to silicone. Morevoer, it was verified that all C. tropicalis were prone to form biofilms on silicone. Regarding C. tropicalis enzymatic activity, it was possible to verify that all isolates were able to express total hemolytic activity on sheep-blood agar medium supplemented with glucose. However, proteinase was only produced by two urine isolates and by the isolates from catheter and blood and only one C. tropicalis (from catheter) was phospholipase positive. All isolates were susceptible to voriconazole, fluconazole and amphotericin B. The largest percentage of susceptibility-dose dependence was observed for itraconazole in 4 strains (57.1%). Furthermore one clinical isolate (14.3%) from urine was found to be resistant to the same compound (MIC = 1 µg/ml). Thus, it is possible to conclude that there was no direct correlation between the virulence factors assayed (secretion of enzymes, adhesion to epithelial cells and silicone and biofilm formation). Concerning C. tropicalis susceptibility, it was not possible to establish any relation with Candida virulence factors as well. However, it is important to highlight that all isolates presented one or more virulence factors

Dendrimers in tissue engineering

Dendrimers are highly branched and multivalent, and monodispersive making them perfect candidates for a myriad of controlled drug delivery applications. Dendrimers possess many other advantages, such as the possibility of modulating surface chemistry and charge, and biodegradation rate and to be processed as scaffolds that can emulate natural extracellular matrices thus opening up unique applications in tissue engineering. The combination of dendrimers and other macromolecules (proteins and carbohydrates), as well as other traditional scaffold polymers, has led to the creation of hybrid scaffolds with new physical, mechanical, and biochemical properties. However, despite the widespread use of dendrimers in biomedical applications, their use in the fabrication of tissue engineering scaffolds remains some-how narrow. The most promising applications of dendritic macromolecules in TE area such as drug delivery strategies, cell differentiation and/or tissue regeneration, 3D/Dynamic platforms and ex vivo/in vivo testing are overviewed and discussed herein.JMO for the financial support provided under the program “Investigador FCT” (IF/01285/2015). MCR acknowledges the IET Institution of Engineering and Technology for the financial support provided under the IET Harvey Research Prize 2017. IMO thanks the financial support under the Norte2020 project (“NORTE-08-5369-FSE-000044”)

Adhesion of non-Candida albicans Candida spp to urinary epithelial cells

Non-Candida albicans Candida (NCAC) spp, are nowadays responsible for more than 50% of Candida infections and it has been shown that the most prevalent NCAC spp are C.tropicalis, C. parapsilosis and C. glabrata. An important step on NCAC spp infection is their ability to adhere to the host tissues. Therefore, the aim of this study was to evaluate the ability of C.tropicalis, C. parapsilosis and C. glabrata to adhere to a urinary epithelial cell line (TCC-SUP). The ability of C.tropicalis ATCC 750, C. parapsilosis ATCC 2201, C. glabrata ATCC 2001 and C. albicans SC5413 to adhere to TCC-SUP cells was evaluated after 2 and 24h. For that, yeast cells (1x107 cell/ml) were incubated with a confluent layer of epithelial cells at 37 ºC and 5 % of CO2. The number of yeast cells adhered to the epithelial cells was determined using an adaptation of the Crystal Violet (CV) staining method. Additionally, the epithelial cell activity was determined by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2- (4-sulfophenyl)-2H-tetrazolium (MTS) viability assay. After 24h of incubation the number of yeasts adhered to epithelial cells was higher than after 2h, unexpectedly except for C. glabrata. Nevertheless, after 24 h, C. glabrata extent of adhesion was still higher than C. tropicalis and C. parapsilosis. This particular behavior of C. glabrata was also observed after 2 h of adhesion, showing a higher number of adhered cells in comparison with the other NCAC spp, which behaved very similarly. The trend observed by CV staining does not seem to be reflected on the metabolic activity of the epithelial cells.The main conclusion of the present work is that NCAC spp are biofilm producers on silicone, and that the adhesion and biofilm formation ability appear to be species and strain dependent

Investigation of Dendrimer-based nanoparticles cellular uptake and cell tracking in a semiautomated microfluidic platform

A microfluidic device such as Kima Pump and Vena8 biochip is able to realize functions that are not easily imaginable in conventional biological analysis, such as highly parallel, sophisticated high-throughput analysis and single-cell analysis in a well-defined manner [1]. Cancer cell tracking within the microfluidic model will be achieved by grafting fluorescent label probe Fluorescein-5(6)-isothiocyanate (FITC) to dendrimer nanoparticles allowing cell visualization by immunofluorescent staining followed by fluorescence microscopy. In this study, synthesis and physicochemical characterization of Carboxymethyl-chitosan/poly(amidoamine) dendrimer nanoparticles (CMCht/PAMAM NPâ s) were performed[2].  Several cancer cell lines such as a HeLA (cervical carcinoma cell line), HTTC-116 (Colon Carcinoma) and Glioblastome cell line (GBM) were exposed to different concentrations of CMCht/PAMAM dendrimer nanoparticles over a period of 7d. After finding the adequate NP concentration, the internalization efficiency was tested, as well as cellular trafficking, in static and dynamic conditions (Kima Pump bioreactor).Portuguese Foundation for Science and Technology (FCT) through the project PEst­C/SAU/LA0026/20