MOLECULAR ANALYSIS OF Candida BIOFILMS

Abstract

The foremost task taken in the present study deals with the selection of Candida species which form biofilm on infected IMDs made up of various biocompatible materials (BCMs). 60% of the clinical samples obtained from the patients with infected indwelling medical devices (IMDs) such as urinary catheters and intra uterine devices showed high incidences of Candida infections. On microscopic examinations these clinically isolated Candida spp. appeared as ovoid, budding yeast, sometimes having mould like hyphae and occasionally with large refractile chalmydospores. Species identification of these strains were performed by both biochemical and microbiological procedures. CHROMagar, a selective media was used as a convenient way for rapid identification of Candida species adhering to medical implanted biocompatible materials. Green, blue, pale pink and pink color colonies were observed for C. albicans, C tropicalis, C. krusei and C. parapsilosis respectively on this selective media. Among different clinical isolates of Candida species, clinical isolate no. AU7 (identified as C. albicans) showed maximum biofilm forming ability on BCM using MTP assay. Maximum dry weight (1.95 mg) for biofilm formed by C. albicans (AU7) was obtained after 48 h at 35°C. Health of the C. albicans biofilm was recorded using MTT assay which was based on reduction of this salt by mitochondrial dehydrogenase to violet color tetrazolium formazan product. Spectrophotometrically reading at 540 nm showed maximum biofilm formation by C. albicans (AU7) take place after 48 h. This correlates with increased cellular density in the biofilm as assessed by electron microscopy techniques (SEM). Data suggests that biofilm were highly metabolically active in its first

24h. However, as the Candida biofilm matures and become more complex (48 to 72 h), the metabolic activity reached its plateau reflecting high number of cells that constitute the mature biofilm. SEM analysis of Candida albicans biofilm topography formed on infected medical devices revealed a dense network of mono or multiplayer of cells embedded within the matrix of extracellular polymer material. Maximum colonization strength of C. albicans biofilm was observed at apH 6.5 and temperature optimum of 35T after 48h using galactose as the carbon source (50mM) in yeast nitrogen base medium. Results from our investigations on temporal development of C. albicans biofilms on polyvinylchloride (PVC) strip showed that biofilms formation on these materials used in manufacture of biocompatible devices progresses in three distinct development phases. Initially, (0-6h) majority of Calbicans cells were present as blastospores (yeast form) adhering to the surface of the strips. At 6-12h C. albicans communities appeared as thick tracks of fungal growth. The intermediate development phase (12-36h) was characterized by the emergence of predominantly noncellular materials, which appeared as hazy-like film covering the C. albicans microcolon^. During the maturation phase (36-72h) the amount of extracellular material increased with incubation time and completely encased within this period. Comparative analysis of C albicans biofilm by static and shaking incubation showed slow shaker speed (5 rpm) to be optimum after 48 hat 35°C when measured using MTT assay. After UV irradiation (96 uJmm-2) for 60s on C. albicans AU7 we were able to select abiofilm deficient mutant BDM, namely- C. albicans bdml on the basis of its rough colonial morphology and its lack ofability to adhere to the MTP wells made up biocompatible material (PVC). (") MTP assay at different time intervals on MTP well showed that maximum crystal violet colorization take places after48 h of growth by C. albicans AU7 in comparison to its biofilm deficient mutant bdml, which showed its incapability for the same. Optimum temperature, pHand NaCl concentration for biofilm deficient mutant bdml were found to be 35°C, 6.5 and 0.5%, respectively. Among different carbon sources tested galactose (50mM) was noticed to be most favorable one. Cell wall extracts of C. albicans (AU7 and its biofilm deficient mutant bdml) obtained by treatment with 2-mercaptoethanol were separated by SDS-polyacrylamide gel electrophoresis and analyzed by western blotting suggests that when the above samples were treated with HRP labeled purified antirabbit IgG (anti-AU7 and mti-bdm7; 1:640 dilution) and developed for substrate color reaction (H202 and 4-chloro-l-napthol) a 58 KDa band as the major antigen present in C. albicans (AU7), which may be responsible for biofilm formation since it was absent in its biofilm deficient mutant form bdml. The in vitro activity of antifungal agents amphotericin B, nystatin, fluconazole and chlorhexidine against pre-formed C. albicans (AU7) and its biofilm deficient mutant (bdml) were assessed using XTT- reduction assay revealed increased susceptibility of biofilm deficient mutant bdml (50% reduction in metabolic activity for the same antifungal at a concentration of 0.25, 1.0, 0.25, 8 (xg/ml respectively) when compared with C. albicans AU7 (50% reduction in metabolic activity for the same antifungal at a concentration of 8, 16, >64, 128 u.g/ml respectively).