Keywords
Candida albicans
Biofilm
Antifungal
How to Cite
Abstract
Candida-associated nosocomial infections are a persistent problem which has been steadily increasing over the years. The emergence of antimicrobial resistant strains has narrowed the spectrum of effective drugs that can be utilised and indicated the need for alternative therapeutics. Garlic is a spice often studied for its unique characteristics. Most of its antimicrobial properties have been attributed to the presence of allicin, which makes up more than 70% of thiosulfinates extracted from garlic. The present study aimed to determine the effects of fresh garlic extract (FGE) towards the expression of two major biofilm genes, Flo-8 and Ndt80 produced by C. albicans that enable the morphogenesis of yeast from planktonic cells to biofilm. Minimum concentration required for FGE to inhibit C. albicans was determined through agar well and disc diffusion assay. Effect of FGE towards preformed C. albicans biofilms was evaluated at ¼× and ½× inhibitory concentrations of FGE and Amphotericin B, respectively by XTT assay. RNA from FGE-treated C. albicans was extracted, reverse transcribed and analysed by Real-Time Polymerase Chain Reaction (RT-PCR) to determine FGE effects towards the expression of Flo-8 and Ndt80. Inhibitory concentration of FGE was determined to be 100 mg/mL. Both genes appeared to be upregulated in the presence of FGE with a higher upregulation noted with Flo-8 (67.34 and 30.20-folds) when compared against Ndt80 (5.55 and 1.79-folds) at 5 mg/mL and 10 mg/mL, respectively. It is surmised that C. albicans upregulated these genes as a survival mechanism in the presence of FGE. The findings from this study indicate that FGE could inhibit C. albicans growth and biofilms. Further research is required to determine the minimum inhibitory concentration (MIC) through broth microdilution and to understand the mechanism behind this phenomenon before it can be utilised as a potential therapeutic to combat Candida-associated nosocomial infections.
References
Branco, J., Martins-Cruz, C., Rodrigues, L., Silva, R. M., Araujo-Gomes, N., Goncalves, T., & Miranda, I. M. (2021). The transcription factor of Ndt80 is a repressor of Candida parapsilosis virulence attributes. Virulence, 12(1), 601-614.
https://doi.org/10.1080/21505594.2021.1878743
Casella, S., Leonardi, M., Melai B., Fratini, F., & Pistelli, L. (2013). The role of diallyl sulfides and dipropyl sulfides in the in vitro antimicrobial activity of the essential oil of garlic, Allium sativum L., and leek, Allium porrum L. Phytotherapy Research, 27(3), 380-383.
https://doi.org/10.1002/ptr.4725
Cavallito, C. J., & Bailey, J. B. (1944). Allicin, the antibacterial principle of Allium sativum; isolation, physical properties and antibacterial action. Journal of the American Chemical Society, 66(11), 1950–1951.
https://doi.org/10.1021/ja01239a048
Dantas, A. D. S., Day, A., Ikeh, M., Kos, I., Achan, B., & Quinn, J. (2015). Oxidative stress responses in the human fungal pathogen, Candida albicans. Biomolecules, 5(1), 142-165.
https://doi.org/10.3390/biom5010142
Fox, E. P., Bui, C.K., Nett, J.E., Hartooni, N., Mui, M. C., Andes, DR, Nobile, C. J., & Johnson, A. D. (2015). An expanded regulatory network temporally controls Candida albicans biofilm formation. Molecular Microbiology, 96(6), 1226-1239.
https://doi.org/10.1111/mmi.13002
Fujisawa, H., Watanabe, K., Suma, K., Origuchi, K., Matsufuji, H., Seki, T., & Ariga, T. (2009). Antibacterial potential of garlic-derived allicin and its cancellation by sulfhydryl compounds. Bioscience, Biotechnology and Biochemistry, 73(9), 1948–1955.
https://doi.org/10.1271/bbb.9009
Jabar, M. A., & Al-Mossawi, A. (2007). Susceptibility of some multiple resistant bacteria to garlic extract. African Journal of Biotechnology, 6(6), 771–776.
Jiang, C., Li, Z., Zhang, L., Tian, Y., Dong, D., & Peng Y. (2016). Significance of hyphae formation in virulence of Candida tropicalis and transcriptomic analysis of hyphal cells. Microbiological Research, 192(2016), 65-72.
https://doi.org/10.1016/j.micres.2016.06.003
Khodavandi, A., Alizadeh, F., Harmal, N. S., Sidik, S. M., Othman, F., Sekawi, Z., Jahromi, M. A. F., Ng, K. P., & Chong, P. P. (2011a). Comparison between efficacy of allicin and fluconazole against Candida albicans in vitro and in a systemic candidiasis mouse model. FEMS Microbiology Letters, 315(2), 87-93.
https://doi.org/10.1111/j.1574-6968.2010.02170.x
Khodavandi, A., Harmal, N. S., Alizadeh, F., Scully, O. J., Sidik, S. M., Othman, F., Sekawi, Z., Ng, K. P., & Chong, P. P. (2011b). Comparison Between allicin and fluconazole in Candida albicans biofilm inhibition and In suppression of HWP1 gene expression. Phytomedicine, 19(1), 56–63.
https://doi.org/10.1016/j.phymed.2011.08.060
Kim, J., & Sudbery, P. (2011). Candida albicans, a major human fungal pathogen. Journal of Microbiology (Seoul, Korea), 49(2), 171–177.
https://doi.org/10.1007/s12275-011-1064-7
Laniado-Laborín, R., & Cabrales-Vargas, M. N. (2009). Amphotericin B: side effects and toxicity. Revista Iberoamericana de Micologia, 26(4), 223-227.
https://doi.org/10.1016/j.riam.2009.06.003
Lemar, K. M., Turner, M. P., & Lloyd, D. (2002). ‘Garlic (Allium sativum) as an anti-Candida agent: A comparison of the efficacy of fresh garlic and freeze-dried extracts. Journal of Applied Microbiology, 93(3), 398-405.
http://doi.org/10.1046/j.1365-2672.2002.01707.x
Li, G., Ma, X., Deng, L., Zhao, X., Wei, Y., Gao, Z., Jia, J., Xu, J., & Sun, C. (2015). Fresh garlic extract enhances the antimicrobial activities of antibiotics on resistant strains in vitro. Jundishapur Journal of Microbiology, 8(5), e14814.
https://doi.org/10.5812/jjm.14814
Liu, J. Y., Li, W. J., Shi, C., Wang, Y., Zhao, Y., & Xiang, M. J. (2015). Mutations in the Flo8 transcription factor contribute to virulence and phenotypic traits in Candida albicans strains. Microbiological Research, 178, 1-8.
https://doi.org/10/1016/j.micres.2015.05.007
Livak, K. J., Schmittgen, T. D. (2001). Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCT Method. Methods, 25, 402-408.
https://doi.org/10.1006/meth.2001.1262
Low, C. F. , Chong, P. P. , Yong, P. V. C, Lim, C. S. Y., Ahmad, Z., & Othman, F. (2008). Inhibition of hyphae formation and SIR2 expression in Candida albicans treated with fresh Allium sativum (garlic) extract. Journal of Applied Microbiology, 105(6), 2169-2177.
https://doi.org/10.1111/j.1365-2672.2008.03912.x
Majewski, M. (2014). Allium sativum: Facts and myths regarding human health. Roczniki Państwowego Zakładu Higieny, 65(1), 1-8.
Mesa-Arango, A. C., Scorzoni, L., & Zaragoza, O. (2012). It only takes one to do many jobs: Amphotericin B as antifungal and immunomodulatory drug. Frontiers in Microbiology, 3, 286.
https://doi.org/10.3389/fmicb.2012.00286
Min, K., Biermann, A., Hogan, D. A., Konopka, J. B. (2018) Genetic analysis of NDT80) family transcription factors in Candida albicans using new CRISPR-Cas9 approaches. mSphere, 3(6), 1-13.
https://doi.org/10.1128/mSphere.00545-18
Nailis, H., Coenye, T., Van Nieuwerburgh, F., Deforce, D., & Nelis, H. J. (2006). Development and evaluation of different normalization strategies for gene expression studies in Candida albicans biofilms by real-time PCR. BMC Molecular Biology, 7, 25.
https://doi.org/10.1186/1471-2199-7-25
Nobile, C. J., Fox, E. P., Nett, J. E., Sorrells, T. R., Mitrovich, Q. M., Hernday, A. D., Tuch, B. B., Andes, D. R., & Johnson, A. D. (2012). A recently evolved transcriptional network controls biofilm development in Candida albicans. Cell, 148(1-2), 126-138. https://doi.org/10.1016/j.cell.2011.10.048
Nobile, C. J., & Johnson, A. D. (2015). Candida albicans Biofilms and Human Disease. Annual Review of Microbiology, 69, 71-92.
https://doi.org/10.1146/annurev-micro-091014-104330
Palacios, D., Dailey, I., Siebert, D., Wilcock, B., & Burke, M. (2011). Synthesis-enabled functional group deletions reveal key underpinnings of Amphotericin B ion channel and antifungal activities. Proceedings of the National Academy of Sciences, 108(17), 6733-6738.
https://doi.org/10.1073/pnas.1015023108
Pierce, C. G., Uppuluri, P., Tristan, A. R., Wormley Jr, F. L., Ramage, G., & Lopez-Ribot, J. L. (2008). A simple and reproducible 96 well plate-based method for the formation of fungal biofilms and its application to antifungal susceptibility testing. Nature Protocols, 3(9), 1494–1500.
https://doi.org/10.1038/nport.2008.141
Rahman, M. S. (2007). Allicin and other functional active components in garlic: Health benefits and bioavailability. International Journal of Food Properties, 10, 245-268.
https://doi.org/10.1080/10942910601113327
Rybak, M. E., Calvey, E. M., & Harnly, J. M. (2004). Quantitative determination of allicin in garlic: Supercritical fluid extraction and standard addition of alliin. Journal of Agricultural and Food Chemistry, 52(4), 682–687.
https://doi.org/10.1021/jf034853x
Salehi, B., Zucca, P., Orhan, I. E., Azzini, E., Adetunji, C. O., Mohammed, S. A., Banerjee, S. K., Sharopov, F., Rigano, D., Sharifi-Rad, J., Armstrong, L., Martorell, M., Sureda, A., Martins, N., Selamoglu, Z., & Ahmad, Z. (2019). Allicin and health: A comprehensive review. Trends in Food Sciences and Technology, 86. 502-516.
https://doi.org/10.1016/j.tifs.2019.03.003
Savastano, C., de Oliveira Silva, E., Gonçalves, L., Nery, J., Silva, N., & Dias, A. (2016). Candida glabrata among Candida spp. from environment health practitioners of a Brazilian Hospital. Brazilian Journal of Microbiology, 47(2), 367-372.
https://doi.org/10.1016/j.bjm.2015.05.001
Shadkchan, Y., Shemesh, E., Mirelman, D., Miron, T., Rabinkov, A., Wilchek, M., & Osherov, N. (2004). Efficacy of allicin, the reactive molecule of garlic, in inhibiting Aspergillus spp. in vitro and in a murine model of disseminated aspergillosis. Journal of Antimicrobial Chemotherapy, 53(5), 832-836.
https://doi.org/10.1093/jac/dkh174
Shuford, J. A., Steckelberg, J. M., & Patel, R. (2005). Effects of fresh garlic extract on Candida albicans biofilms. Antimicrobial Agents and Chemotherapy, 49(1), 471.
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