STUDIES ON EXTRACELLULAR POLYMERIC SUBSTANCES FORMED BY BIOFILM FORMING CANDIDA SPECIES

Abstract

Candida albicans is an opportunistic pathogenic fungus capable of causing a wide variety of infections ranging from superficial to life-threatening systemic infections. Candidiasis is often associated with the formation of biofilms on the surface of inert or biological materials. These biofilms are spatially organized communities of cells encased in a matrix of Extracellular Polymeric Substances (EPS) on a substrate and are particularly characterized by increased resistance to antimicrobials. The present study reports for the isolation and biochemical characterization of EPS produced by a clinical strain of C. albicans isolated from infected clinical samples. About 53.4% of the clinical samples studied showed high incidence of Candida infection, amongwhich C. albicans was found in maximum percentage (41%). Chromogenic agar medium was successfully utilized in the identification of these Candida species from infected clinical samples. On microscopic examination, C albicans appeared as gram positive, ovoid, budding yeast having mould like hyphae and large refractile chlamydospores. Amongst 16 C. albicans isolates, maximum biofilm forming isolate, C. albicans PLV12 was selected on the basis of XTT reduction assay. The assay was used to explore three developmental phases of C. albicans PLV12 biofilm formation. In the first phase (early phase), adherence of candidal cells to the PVC surface takes place; in the second phase (intermediate phase), formation of a matrix with dimorphic switching from yeast to hyphal forms was observed while the last phase (maturation phase) showed an increase in the matrix material (EPS) giving three-dimensional architecture to the biofilm. Quantitative analysis of C. albicans PLV12 EPS showed that biofilms contained significantly reduced content of total carbohydrate (40±8.5%), protein (5.0±1.2%), extracellular DNA (0.2±0.1%) and enhanced amount of glucose (16±3.4%), hexosamine (4.0±1.3%), uronic acid n (0.5±0.1%) and phosphorus (0.7±0.2%) in contrast to its planktonic counterparts. Microscopic examination using SEM, AFM and CLSM revealed that C. albicans PLV12 biofilms have a highly heterogeneous architecture composed of cellular and non-cellular elements with EPS distributed in the cell-surface periphery or at cell-cell interface. The synthesis of EPS during the formation of C albicans PLV12 biofilm was found to be highly dependent on the conditions of incubation and types of substrates used. Under static conditions, EPS synthesis was minimal but was greatly enhanced under mild shaking (15 rpm) conditions. Parameter optimization indicated that maximum EPS yields were achieved at pH 6.3 and temperature 30°C using arabinose as a carbon substrate for growth. Partial characterization of C. albicans PLV12 EPS using chromatographic techniques showed presence of both negatively (D-glucuronic acid) and positively (Nacetylglucosamine) charged components in the exopolysaccharide chain. The major sugars units in the exopolysaccharide chain as revealed by HPLC and GC analysis were found to be glucose, mannose, galactose, rhamnose, N-acetyl glucosamine and D-glucuronic acid. Gel permeation chromatography determined that C. albicans PLV12 produces a heteropolysaccharide having a molecular weight of -300 KD. Structural analysis using FTIR showed presence of [3-glucans ((3 (1—>6) and P (1—>3) and mannans in the exopolysaccharide chain. These were later assigned to a- and P-D-glucose, a-D-mannose, a-L-rhamnose and Nacetyl glucosamine (p-D-GlcNAc) by !H and l3C NMR studies. EMS (3%) treatment showed 19 C. albicans PLVI2 mutants having more than 50% reduction in their biofilm forming ability. Of which, the selected mutant strain,pm4, resulted in 75.2% and 71% reduction in biofilm formation and EPS production respectively, compared with the wild strain. This mutant strain showed bluish-green colonies with rough in morphology on HiChrom Candida agar medium. SEM analysis further explored morphological alterations in biofilms formed by this mutant strain. Comparative analysis of mutant (pm4) EPS with that of wild strain revealed reduction in total carbohydrate, protein and glucose content. The antifungal susceptibility testing indicated increased susceptibility of mutant strain towards Fluconazole, Itraconazole, Ketoconazole and Amphotericin B in contrast to its wild strain. Various control strategies used in the study showed their potential towards C. albicans PLV12 biofilm. Out of which Eucalyptus and Peppermint oils showing 80.87% and 74.16% reduction in C. albicans PLV12 biofilm formation were found to be promising agents. Addition of metabolic inhibitors like DNP and bismuth dimercaprol to the culture medium also resulted in a significant decrease in viable cell counts and EPS yield. Studies with biosurfactant (rhamnolipid) showed 78.6% and 66.79% reduction in biofilm and EPS yield, acting directly on the biofilm matrix to disrupt and solubilize its components and incorporating the matrix into micelles. Thus, prove to be an attractive agent in overcoming C. albicans biofilm-induced resistance. Use of enzymes like Alginate Lyase, Cellulase, Chitinase, Proteinase K, and DNase 1 also revealed a significant decrease in C. albicans PLV12 biofilms by partially degrading matrix material and causing biofilm detachment from the surfaces of the MTP. Interestingly, Lyticase hydrolyzing 0-1, 3 glucan moiety of C. albicans PLV12 EPS resulted in maximum (82%) reduction in biofilms. Further, C. albicans PLV12 biofilms were found to be resistant to almost all the antifungal agents (Fluconazole, Itraconazole, Ketoconazole and Flucytosine) used even at concentrations greatly in excess of their MICs. Only AmphotericinB showed an inhibitory effect on the activity of biofilm cells (62% reduction) but no significant reduction in EPS production was noticed indicating that IV EPS does not contribute a barrier to the penetration of antifungal agents of differing chemical structure. The work presented in the thesis would be helpful in unveiling mystery behind C. albicans polymeric matrix and may provide means to design novel therapies against C. albicans biofilm-based infections.