MOLECULAR CHARACTERIZATION OF BIOFILM FORMING CANDIDA SPECIES ON BIOMATERIALS

Abstract

Biofilms are composed of microbial communities that are attached to a biotic or abiotic surface in which microorganisms usually encase themselves in an extracellular polysaccharide or slime matrix. These biofilms are of serious concern especially in implant associated infections. These infections are believed to be caused by the introduction of pathogenic microorganisms during the implantation or through blood stream infections onto the surface of the newly inserted device. Among them Candida species most notably Candida albicans are the major fungal pathogen of humans and are responsible for significant morbidity and mortality. This dimorphic fungus ranked fourth among biofilm forming agents behind Enterobacteriaceae, Staphylococcus aureus, and Pseudomonas species. C albicans in its yeast form exists as commensal in many healthy human beings, living mainly in the gastrointestinal and vaginal tract, and in the oral cavity. However, it becomes a major pathogen in immunocompromised hosts infected with HIV, or those in intensive care receiving organ transplants. C albicans biofilm formation on biomaterials is a major problem in medical transplants which leads to health risks, and economic loss. Keeping the significance of above facts in view we initiated our research work with the objectives dealing with the molecular analysis of biofilm formed by C albicans on biomaterials used in medically implanted devices. Percentage distribution of fungal pathogen in clinical samples was obtained by plate dilution method. Data showed high incidences of Candida infections (60%). On microscopic examinations these clinically isolated Candida spp. appeared as ovoid, budding yeast, sometimes having mould like hyphae, occasionally large, retractile spores, chlamydospores were also noticed. Species identification of these strains were performed by both biochemical and microbiological procedures. On selective medium CHROMagar clinical isolates of Candida spp. adhering to medically implanted ii biocompatible devices appeared as green, dark violet, blue-gray, lavender and pink colony color for C. albicans, C. glabrata, C. tropicalis, C. parapsilosis and C. krusei respectively out of which C albicans out breaks were found to be maximum. Scanning electron microscopy (SEM) and light microscopic data showed that C. albicans biofilm has sticky dense network of extracellular polysaccharide (EPS). Biochemical and colorimetric analysis data depicted that hexosamine, glucose, mannose, fructose, xylose, arabinose and proteins are major constituents of C albicans biofilm. XTT (2, 3-bis [2-Methoxy-4-nitro-5-sulfophenyl]- 2H-tetrazolium-5-carboxanilide) reduction assay data showed maximum adherence with 48hr grown culture. Data also suggested 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. Maximum colonization strength of C albicans biofilm was observed at a pH 6.5 and temperature optimum of 37°C after 48h. The in vitro activity of clinically used antifungal agents fluconazole, ketoconazole, clotrimazole, itraconazole, nystatin, caspofungin, variconazole and amphotericin B against pre-formed C albicans biofilms was assessed using the XTT-reduction assay. Experiments revealed that activity of nystatin, caspofungin and other azole derivative like ketoconazole, clotrimazole, itraconazole, variconazole against biofilms was reduced about 27, 11, 100, 3, 2, >250 times respectively compared with their activities against planktonic cultures. Importantly, complete killing of cells within the biofilms was never achieved, as reflected by residual metabolic activity of biofilms. Studies were undertaken to analyze C. albicans biofilm on commonly used biocompatible biomaterial such as polypropylene, polystyrene, polyvinylchloride, silicone rubber, polyacrylamide and steel. These biofilm were analyzed by Confocal laser scanning microscopy (CLSM) using fluorescent staining with Propidium iodide (PI; 8 uM) for biofilm in residing Candida cells and fluorescein isothiocynate-concanavalin A (FITC-ConA; 50 ug/ml) to visualize EPS. Biofilm thickness analysis was done by z-sectioning for each biomaterial revealed that number of microorganisms and EPS produced on different layers varies with biomaterials. Green (FITC-Con A) and Red (PI) fluorescent intensity graph and XTT tetrazolium reduction assay showed differential expression pattern in term of metabolic activity and thickness of biofilm. Quantitative CLSM studies revealed that polymer surface properties and chemical interactions affects adherence and hence biofilm development. Biomaterial surface hydrophobicity and roughness was studied by Goniometric analysis and Atomic force microscopy (AFM) respectively. Data revealed that among all the biomaterials selected PVC was found to possess highest hydrophobicity (97°), roughness (134nm), and colonization (117.5 urn biofilm thickness). Information retrieve from Candida Genome Database (CGD) shows that multiple genes are responsible for C. albicans biofilm regulation. During the biofilm formation induction of drug efflux pumps get induced and this make the biofilm more resistant towards antifungal therapy, but role of these pumps during biofilm formation remain unexplored, keeping the fact in view role of Candida ABC transporter (cdrl) on biofilm formation was also analyzed. Cloning and characterization of CDR1 gene (CandidaABC transporter) of biofilm forming C. albicans was done in to vector pSKPDR5PPUS at Spel site and transformed to Sachcaromyces cereviceae host ADl-8u" (MATapdrl-3 hisl ura3 Ayorlr.hisG Asnq2::hisG Apdr5::hisG ApdrlO::hisG Apdrllr.hisG Aycflr.hisG ApdrSMisG Apdrl5::hisG) by lithium acetate transformation method. The cloned product was designated as Apcdrl. Cloning was confirmed through drug susceptibility tests, PCR, and SDS-PAGE which gave overexpressed protein (cdrlp) band of 170kd. Role of CDR1 gene in biofilm formation was tested by studying effect of different metals like Copper, Zinc, Magnesium, Phosphorus, Manganese, and Cadmium on biofilm iv formed by Apcdrl with its control along with drug susceptibility tests. Apcdrl was shown to be hypersensitive towards azole drugs and have demonstrated its inability to form complete biofilms under metal stress condition thereby depicting the involvement of drug efflux pump towards biofilm formation. Prevention and control of C. albicans biofilm achieved by using rhamnolipids, plant oils, enzymes and silver coating. Rhamnolipid (8 ug or 4% v/v concentration) isolated from Pseudomonas aeruginosa by acid precipitation technique was able to reduce 91%, 86%, 85%, and 68% of biofilm formed on silicone rubber, polystyrene, polyvinylchloride, and polypropylene respectively. The inhibitory effect of 30 plant oils (almond, alsi, babchi, babuna, cade, castor, chaulmoogra, clove, coconut, eucalyptus, ginger grass, ginger, jasmine, jojoba, juniper, jyotishmati, khus, lavender, mahua, malkangani, musturd, neem, ocimum, peppermint, rose, tea tree, til, tulsi, walnut and wheatgerm) was evaluated against C. albicans by standard disc diffusion assay, eighteen (babchi, castor, clove, coconut, eucalyptus, ginger grass, ginger, jasmine, juniper, lavender, mahua, malkangani, musturd, ocimum, peppermint, rose, tea tree, and tulsi ) among the thirty plant oils selected were found to be effective. The Minimum Inhibitory Concentrations (MICs) values were calculated by agardilution and brothmacro dilution assay respectively. Effect of these oils was further evaluated against C. albicans biofilm results depicted that eucalyptus, peppermint, ginger grass, and clove oils can reduce 80.87, 74.16%, 40.46% and 28.57% biofilm respectively. Enzymatic activities of polygalactouronse, pectin lyase, cellulase, arabinase, alginate lyase, proteinase and chitinase were used against C. albicans biofilm taking fluconazole as positive control. Data showed maximum 70.7% reduction in C. albicans biofilm with alginate lyase while 66.6%, 49.4%, 33.2%, 29.6%, 19%, 14.8%, and 13.3%% reduction was achieved by pectin lyase, cellulase, chitinase, polygalactouronse, arabinase, proteinase glucose oxidase, and proteinase respectively. C. albicans biofilm inhibition on PVC surface was studied by silver-coating for different time intervals of 5,10,15,20 and 25 sec. followed by FE-SEM, AFM and goniometric analysis. Data showed that 15 sec of silver coating was sufficient to complete prevention of C. albicans biofilm on PVC surface. Work presented in this thesis may prove very useful to combat with Candida albicans biofilm related infections.