Azeredo, Joana

Henriques, Mariana

Negri, M.

Oliveira, Rosário

Universidade do Minho: RepositoriUM

Semana de Engenharia 2010 Guimarães, 11 a 15 de Outubro  EFFECT OF CANDIDA TROPICALIS IN PLANKTONIC AND BIOFILM FORM ON URINARY EPITHELIAL CELLS Melyssa Negri, Mariana Henriques, Joana Azeredo, Rosário Oliveira IBB-Institute for Biotechnology and Bioengineering, Centre of Biological Engineering, Universidade do Minho, Campus de Gualtar 4710-057, Braga, Portugal E-mail: melyssanegri@deb.uminho.pt     KEYWORDS Candida tropicalis; epithelial cells; biofilm; adhesion.  INTRODUCTION Urinary tract infections (UTI) are usually the most common type of hospital acquired infection in developed countries. Although medical devices are indispensable in the management of critically ill patients, about 20% of fungal UTI are associated to the use of urinary catheters. Candida species are the most frequently isolated fungi, corresponding to approximately 80% of fungal associated nosocomial infections and are the second most common species responsible for patients' mortality. 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 being responsible for a high rate of patients’ mortality. Adhesion to host surfaces (epithelial cells and medical devices), as well as biofilm formation, are considered the first step to initiate Candida infection. Hence, the colonization of indwelling devices like urinary catheters by C. tropicalis poses a critical problem. Therefore, more knowledge has to be acquired in order to understand and prevent the formation of these biofilm infections.  Objective The aim of this study was to investigate the influence of C. tropicalis growth form (planktonic or biofilm) in its adhesion to TCC-SUP cells (human urinary bladder).  MATERIALS AND METHODS This study was conducted with one isolate of C. tropicalis obtained from a patient with candiduria admitted to the intensive care unit at the University Hospital in Maringá, Paraná, Brazil and C. tropicalis ATCC 750 was also used, as a control. Adhesion assays were performed incubating one silicone cupon with pre-formed C. tropicalis 24h biofilm or 1 ml of C. tropicalis cell suspension (1.0 x 107 cells/mL), at 37ºC, on a confluent layer of epithelial cells. The extent of adhesion was evaluated after 2 h of incubation using an adaptation of the crystal violet staining method. Moreover, cell viability was also assessed, after contact with yeasts, either by trypan blue staining and using 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) viability assay. Samples were also observed under scanning electron microscopy (SEM).  RESULTS From the results obtained it was possible to verify that, in general, Candida cells adhered to epithelium (Fig 1). Furthermore, the clinical isolate biofilm cells adhered in higher extent than planktonic cells. Nevertheless, comparing both strains, it can be highlighted that the reference strain grown planktonically adhered significantly more (p<0.05) to epithelial cells than C. tropicalis from candiduria, which was confirmed through ultra structure analysis by SEM (Fig. 1).  C. tropicalis in biofilm form caused higher epithelial cells death than their planktonic counterparts (Fig. 2). Moreover, epithelial cells showed less metabolic activity when in contact with biofilms.  (I)  0,0000,0200,0400,0600,0800,1000,120BCT750 BCT69 CT750 CT69Abs(590nm)/cm2 (II)  Figures 1: (I) C. tropicalis extent of adhesion SUP cells after 2 hours of incubation crystal violet staining expressed as absorbance/cm² (Abs/cm²). BCT750 - C. tropicalis ATCC 750 biofilm; BCT69 - C. tropicalis clinical isolate biofilmC. tropicalis ATCC 750 plantonic; CT69 clinical isolate plantonic. (II) SEM of yeasts adhered to TCC-SUP:. A) C. tropicalis from candiduriatropicalis ATCC 750.   Figures 2: Death of the cells of the epithelial cell (TCCSUP). The cell number was quantified by Trypan blue after 2h of incubation to TCC-SUP cellsepithelial cell TCC-SUP; CS - silicone coupon; BCT750 - C. tropicalis ATCC 750 biofilm; BCT69 tropicalis from candiduria biofilm; CT750 tropicalis ATCC 750; CT69 - C. tropicaliscandiduria.  CONCLUSIONS Thus, it is possible to conclude that C. tropicalisable to cause more epithelial cell death when in biofilm  Semana de Engenharia 2010 Guimarães, 11 a 15 de Outubro  to TCC-measured by ; CT750 - - C. tropicalis  and B) C.  -. CEU - - C. - C.  from  were form. This highlights the importance of biofilm formation, associated to the use of urinary catheters, on C. tropicalis virulence.  AUTOBIOGRAPHIES  MELYSSA NEGRI graduated in University Pontifícia Católica of Paraná2002. She obtained her masteron pathogenic yeast, in 2006Paulo, Brazil. She is working on Professor Rosário Oliveira’s group, performing her PhD ongene expression of virulence factors: correlation with biofilms and adhesion of Candida tropiccells. Her e-mail is melyssanegri@deb.uminho.pther web page is   http://www.ceb.uminho.pt/pessoas/pid.aspx?id=200 MARIANA HENRIQUES Engineering in 1998 at University of Minho. She then post-graduated in Industrial Engineering in 2000 and obtained her PhD degree in Chemical and Biological Engineering in 2005 at the University of Minho. She is currently an invited assistant at the Department of Biological Engineering at University of Minho, and her main research interests are Candidafactors, monoclonal antibodies production processes, and characterization of biomaterials. Her eis mcrh@deb.uminho.pt and her web page can be found at http://www.deb.uminho.pt/pessoas/docente.aspx?id=28.  ROSÁRIO OLIVEIRA Engineering at University of Luanda, in 1975. She obtained her PhD degree in Engineering Science in 1991 at the University of Minho. She is currently a full professor at the Department of Biological Engineering of the University of Minho. She is the head of the master degree in Environmental Management and head of the Biofilm research group. Her main research interests are in microbiology and environmental sciences, and animal cell culture technology. Her eis roliveira@deb.uminho.pt http://www.deb.uminho.pt/pessoas/en/docente.aspx?id=36. Biology Science at , Brazil, in  degree in Microbiology  at the University of São  analysis of alis to epithelial  and . graduated in Biological  species virulence -mail address  graduated in Chemical -mail and her web page is 

Effect of Candida tropicalis in planktonic and biofilm form on urinary epithelial cells

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