The Evaluation of the virulence factors of clinical Candida isolates and the anti-biofilm activity of Elettaria cardamomum against multi-drug resistant Candida albicans


1 Vivekanandha College of Arts and Sciences for Women (Autonomous), Tamil Nadu, India

2 Department of Medical Laboratory Sciences, College of Medicine and Health Sciences, Hawassa University, Ethiopia


Background and Purpose: Today, treatment of life-threatening fungal infections, caused by Candida species, has become a major problem. In the present study, we aimed to evaluate the antifungal susceptibility patterns of different clinical Candida isolates, determine the virulence factors in multi-drug resistant (MDR) Candida species, and assess the anti-biofilm activity of Elettaria cardamomum against MDR Candida species.
Materials and Methods: A total of 202 isolates from different Candida species were obtained from three governmental hospitals in Senthamangalam, Tiruchengode, and Namakkal, Tamil Nadu, India. The isolates were identified, using conventional methods. Candida species were tested for virulence factors such as biofilm, protease, and phospholipase activity. The minimum inhibitory concentration (MIC) of Elettaria cardamomum against MDR biofilm-forming C. albicans was determined, using plate and tube methods.
Results:The identified Candida isolates (n=202) were C. albicans (74/202), C. glabrata (53/202), C. parapsilosis (44/202), C. tropicalis (15/202), and C. dubliniensis (16/202). The isolates were subjected to antifungal susceptibility testing and the virulence factors were determined. In terms of biofilm production, non-C. albicans species such as C. dubliniensis showed 75% activity. Also, regarding protease activity, C. parapsilosis (75%) showed the highest percentage of protease production. In addition, Candida species showed strong positivity for phospholipase activity (62.87%). In the MIC method, the acetonic extract completely inhibited biofilm production at a concentration of 125 μl (56.25 μg). In comparison with the ethanolic extract, the acetonic extract showed major activity against biofilm production.
Conclusion: Based on the findings, pathogenic C. albicans species were inhibited by the ethanolic and acetonic extracts of
E. cardamomum. In recent years, MDR and biofilm-forming pathogenic Candida species have been increasingly detected
in clinical settings. Therefore, herbal derivatives might contribute to the treatment of infections without causing any sideeffects
and prevent the associated mortality.


1. Ramage G, Bachmann S, Patterson TF, Wickes BL, Lopez-Ribot JL. Investigation of multidrug efflux pumps in relation to fluconazole resistance in Candida albicans biofilms. J Antimicrob Chemother. 2002; 49(6):973-80.
2. Sullivan D, Coleman D. Candida dubliniensis: an emerging opportunistic pathogen. Curr Top Med Mycol. 1997; 8(1-2):15-25.
3. Davies J. Inactivation of antibiotic and the dissemination of resistance genes. Science. 1994; 264(5157):375-82.
4. Bauer AW, Kirby WM, Sherris JC, Turck M. Antibiotic susceptibility testing by standardized single disc method. Am J Clin Pathol. 1996; 45(4):493-6.
5. Antonio D, Romani F, Pontieri E, Fioritoni G, Caracciolo C, Bianchini S, et al. Catheter-related candidaemia caused by candida lipolytica in a patient receiving allogenic bone marrow transplantation. J Clin Microbiol. 2002; 40(4):1381-6.
6. Baillie GS, Douglas LJ. Role of dimorphism in the development of Candida albicans biofilms. J Med Microbiol. 1999; 48(7):671-9.
7. Mane A, Kulkarni A, Risbud A. Biofilm production in oral Candida isolates from HIV-positive individuals from Pune, India. Mycoses. 2013; 56(2):182-6.
8. Yigit N, Aktas E, Dagistan S, Ayyildiz A. Investigating biofilm production, coagulase and haemolytic activity in Candida species isolated from denture stomatitis patients. Eur J Med. 2011; 43(1):27-32.
9. Arora DS, Kaur GJ. Antibacterial activity of some Indian medicinal plants. J Nat Med. 2007; 61(3):313-7.
10. Dhulap S, Anita M, Hirwani RR. Phytopharmacology of Elettaria cardamomum. Pharm Rev. 2008; 2(4):27-35.
11. Parthasarathy VA, Chempakam B, Zachariah TJ. Chemistry of spices. Wallingford: CAB International; 2008.
12. Agaoglu S, Dostbil N, Alemdar S. Antimicrobial effect of seed extract of cardamom (Elettarıa cardamomum Maton). YYU Vet Fak Derg. 2005; 16(2):477-80.
13. Kaushik P, Goyal P, Chauhan A, Chauhan G. In Vitro Evaluation of Antibacterial Potential of Dry FruitExtracts of Elettaria cardamomum Maton (Chhoti Elaichi). Iran J Pharm Res. 2010; 9(3):287-92.
14. Wayne PA. Zone diameter interpretive standards, corresponding minimal inhibitory concentration (MIC) interpretive breakpoints, and quality control limits for antifungal disk diffusion susceptibility testing of yeasts. Third International Supplement CLSI document- M444-S3, New York, US; 2009.
15. Yigit N, Aktas E, Dagistan S, Ayyildiz A. Investigating biofilm production, Coagulase and Hemolytic activity in Candida species isolated from denture stomatitis patients. Eurasian J Med. 2011; 43(1):27-32.
16. Gokce G, Cerikcioglu N, Yagci A. Acid proteinase, phospholipase and biofilm production of Candida species isolated from blood cultures. Mycopathologia. 2007; 164(6):265-9.
17. Samaranayake LP, Raeside JM, Macfarlane TW. Factors affecting the phospholipase activity of Candida species by invitro. Sabouraudia. 1984; 22(3):201-7.
18. Rosenberg M, Gutnick D, Rosenberg E. Adherence of bacteria to hydrocarbons: a simple method for measuring cell-surface hydrophobicity. FEMS Microbiol Let. 1980; 9(1):29-33.
19. Le Grand A, Wondergem PA, Verpoorte R, Pousset JL. Anti-infectious phytotherapies of the treesavannah of Senegal (West-Africa) II. Antimicrobial activity of 33 species. J Ethnopharmacol. 1988; 22(1):25-31.
20. Branchini ML, Pfaller MA, Rhine-Chalberg J, Frempong T, Isenberg HD. Genotypic variation and slime production among blood isolates and catheter isolates of Candida parapsilosis. J Clin Microbiol. 1994; 32(2):452-6.
21. Segal E, Clad D. Candida species and Blastoschizomyces capitus. In: Ajello L, Hay RJ, editors. Topley & Wilson’s microbiology and microbial infections. 9th ed. London: Arnold, Hodder Headline; 1998. 423.
22. Cox GM, Perfect JR. Fungal infections. Curr Opin Infect Dis. 1993; 6:422-6.
23. White TC, Holleman S, Dy F, Mirels LF, Stevens DA. Resistance mechanism in clinical isolates of Candida albicans. Antimicrob Agents Chemother. 2002; 46(6):1704-13.
24. Andriole VT. The 1998 Garrod lecture. Current and future antifungal therapy: new targets for antifungal agents. J Antimicrob Chemother. 1999; 44(2):151-61.
25. Wynn RL, Jabra-Rizk MA, Meiller TF. Fungal drug resistance, biofilms, and new antifungals. Gen Dent. 2003; 51(2):94-8.
26. Jabra-Rizk MA, Falkler WA, Meiller TF. Fungal biofilms and drug resistance. J Emerg Infect Dis. 2004; 10(1):14-9.
27. Dos Santos Abrantes PM, McArthur CP, Africa CW. Multi-drug resistant (MDR) oral Candida species isolated from HIV-positive patients in South Africa and Cameroon. Diagn Microbiol Infect Dis. 2014; 79(2):222-7.
28. Badiee P, Badali H, Diba K, Ghadimi Moghadam A, Hosseininasab A, Jafarian H, et al. Susceptibility pattern of Candida albicans isolated from Iranian patients to antifungal agents. Curr Med Mycol. 2016; 2(1):24-9.
29. Inci M, Atalay MA, Koç AN, Yula E, Evirgen O, Durmaz S, et al. Investigating virulence factors of clinical Candida isolates in relation to atmospheric conditions and genotype. Turk J Med Sci. 2012; 42(2):1476-83.
30. Tumbarello M, Posteraro B, Trecarichi EM, Fiori B, Rossi M, Porta R, et al. Biofilm production by Candida species and in adequate antifungal therapy as predictors of mortality for patients with Candidemia. J Clin Microbiol. 2007; 45(6):1843-50.
31. Jasim ST, Flayyih MT, Hassan AA. Isolation and identification of Candida spp. from different clinical specimens and study the virulence factors. World J Pharm Pharmaceut Sci. 2016; 5(7):121-37.
32. Dan M, Poch F, Levin D. High rate of vaginal infection caused by non-C. albicans Candida species among asymptomatic women. Med Mycol. 2002; 40(4):383-6.
33. Sachin CD, Ruchi K, Santosh S. In vitro evaluation of proteinase, phospholipase and haemolysin activities of Candida species isolated from clinical specimens. Int J Med Biomed Res. 2012; 1(2):153-7.
34. Tsang CS, Chu FC, Leung WK, Jin LJ, Samaranayake LP, Siu SC. Phospholipase, proteinase and haemolytic activities of Candida albicans isolated from oral cavities of patients with type 2 diabetes mellitus. J Med Microbiol. 2007; 56(10):1393-8.
35. Deorukhkar S, Saini S. Evaluation of phospholipase activity in biofilm forming Candida species isolated from intensive care unit patients. Br Microbiol Res J. 2013; 3(3):440-7.
36. Emira N, Mejdi S, Dorra K, Amina B, Eulogio V. Comparison of the adhesion ability of Candida albicans strains to biotic and abiotic surfaces. Afr J Biotechnol. 2011; 10(6):977-85.
37. Eggimann P, Garbino J, Pittet D. Epidemiology of Candida species infections in critically ill nonimmunosuppressed patients. Lancet Infect Dis. 2003; 3(11):685-702.
38. Anonynous A. World Health Organization summary of WHO guidelines for the assessment of herbal medicines. Gram. 1993; 28:13-4.
39. Baker JT, Borris RP, Carte B, Cordell GA, Soejarto DD, Cragg GM, et al. Natural product drug discovery and development: New perspective on international collaboration. J Nat Prod. 1995; 58(9):1325-57.
40. Al-Saimary IE, Bakr SS, Khudaier BY, Abbas YA. Efficiency of antibacterial agents extracted from Thymus vulgaris I. (Lamiaceae). Int J Nutr Wellness. 2007; 4:1.
41. Aneja KR, Sharma C. Antimicrobial potential of fruit extracts of Elettaria cardamomum maton (chhoti elaichi) against the pathogens Causing ear infection. Pharmacologyonline. 2010; 3:750-6.