Year: 2011
Type: dissertation
Abstract: Candida tropicalis is a common species related to nosocomial infections, namely candidemia and candiduria. Several virulence factors seem to be responsible for C. tropicalis infections, which present high potential for dissemination and mortality. Adhesion to surfaces (medical devices and host cells) and biofilm formation, are considered important factors that contribute to the development of candidosis. Hence, the colonization of indwelling devices like urinary catheters by C. tropicalis poses a critical problem. Further, adhesion and invasion of host cells by C. tropicalis is considered the first step to initiate systemic infections. Once adhered to epithelium, C. tropicalis are able to secrete hydrolytic enzymes that cause damage in host cells membrane integrity, leading to dysfunction or disruption of host structures. Thus, the main aim of this work was to characterize the virulence factors of C. tropicalis as well as to evaluate adhesion to biotic and abiotic surfaces, biofilm formation, expression of hydrolytic enzymes and antifungal susceptibility of C. tropicalis clinical isolates from urine and blood cultures and from central venous catheters. Accordingly, in order to enhance the knowledge in the process of C. tropicalis adhesion and consequent biofilm formation in urinary catheters, the first goal of this research was to develop an in vitro dynamic model, with silicone and latex urinary catheters, using artificial urine (AU). Moreover, Candida surface hydrophobicity was also evaluated, as well as the biofilm matrix content in terms of proteins and carbohydrates. So, this model using AU was shown to be suitable for studies mimicking the real body conditions. Additionally, C. tropicalis was, in fact, able to colonize both urinary catheters in the presence of AU and to detach from these catheters and move against the flow, demonstrating their ability to colonize distal sites.In vitro studies for the assessment of yeast cells adhesion capability to host tissues are essential to characterise the virulence of Candida species. However, the assessment of the number of adhered yeast cells by traditional methods is time consuming. Therefore, a simple methodology, using crystal violet staining, was developed to quantify in vitro adhesion of different Candida species to epithelial cells. The method was validated for the different Candida reference strains of different species by comparison with traditional microscope observation and enumeration. The proposed technique is easy to perform and reproducible, enabling the determination of adhesion ability of Candida species to an epithelial cell line. After standardizing the methodologies to evaluate Candida adhesion ability, the next step was the characterization of C. tropicalis virulence, by assessing antifungal susceptibility and comparing the expression of several virulence factors. Regarding adhesion, it can be highlighted that C. tropicalis strains adhered in significantly higher number to epithelium than to silicone. Furthermore, all C. tropicalis strains were able to form biofilms and to express total haemolytic activity. However, protease and phospholipase positive response were detected only in few isolates but from different sites of isolation. All isolates were susceptible to voriconazole, fluconazole and amphotericin B. Four strains were susceptible-‐dose dependent to itraconazole and one clinical isolate was found to be resistant to this agent. Then, it was investigated the interaction of C. tropicalis with three different human cell lines: TCC-‐SUP (epithelial cells from urinary bladder); HeLa (epithelial cells from cervical carcinoma) and Caco-‐2 (epithelial cells from colorectal adenocarcinoma). Specifically, the degree of human cells damage and activity reduction induced by C. tropicalis adhesion and the role of Candida tropicalis aspartyl proteinases (SAPT) genes expression in this process were assessed. It was possible to observed that C. tropicalis strains were able to adhere to the different human cells, although, in a strain and cell dependent manner. Concerning human cells response to C. tropicalis, the highest cell activity inhibition was obtained for Caco-‐2, followed by TCC-‐SUP and HeLa cells. C. tropicalis strains in contact with the different types of epithelial cells exhibited a wide range of expression profiles of SAPT genes, however, SAPT3 was the gene expressed in a higher level. Finally, it was studied the behaviour of C. tropicalis in biofilms of different ages (24-‐120 h) formed in artificial urine (AU) and their effect in human urinary bladder cells (TCC-‐SUP). A similar profile in metabolic activity along biofilm age was found among strains, with an increase from 72 to 96 h and a decrease from 96 to 120 h. Candida tropicalis biofilm cells were able to adhere to TCC-‐SUP cells, in general, independently of biofilm age. Yeasts affected TCC-‐SUP cells, with difference among biofilms and strains. Generally, SAPT3 was highly expressed in comparison with other SAPT genes. In summary, C. tropicalis strains were able to form biofilms in AU, in static or dynamic mode, although, with differences among strains. It is important to emphasize that human cells response to C. tropicalis adhesion, as well as SAPs production, is strain and epithelial cell line dependent. Additionally, it should be highlighted that C. tropicalis cells detached from biofilms are able to colonize human cells and cause some injury and reduction of metabolic activity. Generally, SAPT3 was highly expressed compared to other SAPT genes.
Language: English
Subfield: Infectious Diseases
Field: Medicine
Domain: Health Sciences
Open Access status: green
OpenAlex ID: W2248415420