Candida albicans biofilms on artificial surface in contact with innate immune cells.

ARCE MIRANDA JE; BARONETTI, JOSÉ L; BOTIGLIERI, M; SOTOMAYOR, C; ALBESA, I; PARAJE, M GABRIELA.

Córdoba

Congreso; 1° Reunión Internacional de Ciencias Farmacéuticas RICIFA.; 2010

Organizado por el Dto. de Farmacia, Facultad de Ciencias Químicas, Universidad Nacional de Córdoba y el Dto. de Farmacia, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario.

Introduction It has been documented that at least 65% of all microbial infections are related to formation of biofilms. They are structured communities encased in a matrix of exopolymeric substances, display unique characteristics that confer survival advantages over their planktonic counterparts. Many types of yeast such as Candida and Cryptococcus have been associated with the presence of biofilms. The formation of these organizations by Candida primarily begin with adherence and colonization of a biotic host or an artificial surface as for example medical devices in current use, including stents, shunts, prostheses (voice box, heart valves, dentures, etc.), implants (lens, breast, etc.), and various types of catheters (1). We previously developed a model of Candida biofilms attached to polystyrene microtiter plates, where the oxidative metabolism under different culture conditions was assayed. In the present study, the effect of macrophages (Mø), a very important cell of the immune system, on the production of reactive oxygen species (ROS), the liberation of nitric oxide (NO), and the activity of superoxide dismutase (SOD) in these cultures was evaluated.   Materials and methods Candida albicans was incubated in a 96-well polypropylene microtiter plate to form mature biofilms. Then, mouse macrophage cell line (RAW) or medium alone was aggregated to these plates for 1 h and the supernatants were obtained by centrifugation. The biofilms-forming ability was measured by determination of the adhesion to polystyrene microtiter plates (2), which is based on the ability of fungi to form biofilms on solid surfaces and uses CV to stain biofilms. The Biofilms Biomass Unit (BBU) was arbitrarily defined with 0.1 OD595 equal to 1 BBU (3). The extracellular production of ROS was detected by the reduction of nitro blue tetrazolium to nitroblue diformazan. The supernatant was separated by measuring the extracellular production of ROS (eROS). The reaction was proportional to the ROS generated in biofilm and was measured by OD at 540 nm (3). The NO production was evaluated as nitrite by a microplate assay method using the Griess reagent. Absorbance was measured at 540 nm in a microplate reader and results were expressed as mM (3). Total SOD activity was assayed photochemically based on the inhibition of nitro blue tetrazolium (NBT) reduction (4).   Results The aggregated of Mø to the cultures results in a decrease in the biomass of mature biofilms with respect to control cultures (medium alone). In addition, we were not able to detect the production of eROS and NO by Candida biofilms subjected to Mø or medium alone. On the other hand, the SOD activity of bifofilms co-cultured with Mø was higher than that found in culture controls.   Conclusions Our results show that Mø are able to eradicate mature biofilms from a polymeric surface. Furthermore, the presence of these cells also induces a strong antioxidative profile in our model, indicated by the high SOD activity found in this system. In this way, this antioxidant power could be the reason for which the production of eROS and NO was not detectable. Therefore, in this in vitro model, Mø can interact with biofiloms already formed, diminishing their biomass and altering its oxidative balance, however, futures studies are necessary to explain the mechanism by which these cells eradicate the biofilms.   Acknowledgments The authors wish to thank Bioq. Paula Icely. This work was supported by the following Grants: FONCyT, CONICET and SECyT.   Reference 1.      Seneviratne CJ, Jin LJ, Samaranayake YH, Samaranayake LP. Cell Density and Cell Aging as Factors Modulating Antifungal Resistance of Candida albicans Biofilms. Antimicrobial Agents and Chemotherapy. 2008; p. 3259–3266 2.      O'Toole GA, Kolter R. Initiation of biofilm formation in Pseudomonas fluorescens WCS365 proceeds via multiple, convergent signaling pathways: a genetic analysis. Mol.Microbiol. 1998; 28:449–461. Zernotti ME, Angel Villegas N, Roques Revol M, Baena-Cagnani CE, Arce Miranda JE, Paredes ME, et al. Bacterial biofilm evidence in nasal polyposis. J Investig Allergol Clin Immunol. 2010. In press. Beauchamp C, Fridovich I. Superoxide dismutase: improved assays and an assay applicable to acrylamide gels. Anal Biochem. 1971; 44(1):276-287.