In Vitro Evaluation of Biofilm Forming Capacity and Antifungal Resistance of Yeast Isolated from Individuals Aged 18-25 Years

Yıl 2023, , 1 - 18, 21.07.2023

Gülçin Özcan Ateş , Müşerref Otkun

https://doi.org/10.33484/sinopfbd.1310745

Öz

Microbiota studies are an increasingly important issue today. In the literature, there are limited studies on the antifungal resistance and biofilm formation capacity of yeasts isolated from the mouths of young individuals. For this reason, our study was carried out with 133 yeast isolates isolated from the mouths of 17 young individuals between the ages of 18-25 in 2018-2020. When the biofilm-forming capacities of 133 isolates were examined, it was determined that 99.25% were biofilm producers by tissue culture plate method and 66.92% by tube method. One hundred thirty-three yeast isolates and seven reference strains were first evaluated against fluconazole antifungal by agar disc diffusion method. The isolates were found to be susceptible to fluconazole. According to this result, 20 isolates with strong biofilm-forming capacity were selected from 133 yeast isolates. Antifungal resistance was evaluated with fluconazole, itraconazole, clotrimazole, amphotericin B and nystatin gradient test strips. It was determined that 20 isolates were resistant to amphotericin B, and 18 were resistant to nystatin antifungal. It was determined that the sensitivity of itraconazole to 14 isolates and clotrimazole to 3 isolates was dose-dependent. As a result, azole group antifungals can be used mainly in treating oral yeast infections.

Anahtar Kelimeler

Antifungal resistance, Biofilm, Candida, Gradient test, Oral yeast.

Destekleyen Kurum

Çanakkale Onsekiz Mart üniversitesi Bilimsel Araştırma Projeleri Koordinasyon Birimi

Proje Numarası

THD-2020-3352

Teşekkür

We would like to thank Prof. Dr. Beyza ENER for the supply of reference culture (Candida albicans ATCC 10231, C. albicans ATCC 14053, C. albicans ATCC 24433, Candida tropicalis ATCC 1021, Candida parapsilosis ATCC 22019, Cryptococcus neoformans ATCC 90112). This study was conducted at Çanakkale Onsekiz Mart University Experimental Research Center (ÇOMÜDAM) and Çanakkale Onsekiz Mart University Faculty of Medicine Medical Microbiology Laboratory.

18-25 Yaş Arası Bireylerden İzole Edilen Mayaların Biyofilm Oluşturma Kapasitesinin ve Antifungal Direncinin İn Vitro Olarak Değerlendirilmesi

Öz

Mikrobiyota çalışmaları günümüzde önemi giderek artan bir konudur. Literatürde genç bireylerin ağızlarından izole edilen mayaların antifungal direnci ve biyofilm oluşturma kapasitesi üzerine sınırlı sayıda çalışma bulunmaktadır. Bu nedenle çalışma 2018-2020 yıllarında 18-25 yaş arası 17 genç bireyin ağzından izole edilen 133 maya izolatı ile gerçekleştirilmiştir. 133 izolatın biyofilm oluşturma kapasiteleri incelendiğinde doku kültürü plak yöntemi ile %99.25'inin, tüp yöntemi ile %66.92'sinin biyofilm oluşturduğu belirlendi. 133 maya izolatı ve 7 referans suş ilk önce agar disk difüzyon yöntemiyle flukonazol antifungaline karşı değerlendirildi. İzolatların flukonazole duyarlı olduğu bulundu. Bu sonuca göre 133 maya izolatı arasından biyofilm oluşturma kapasitesi güçlü 20 izolat antifungal dirençliliğin belirlenmesi için seçilmiştir. Antifungal direnç flukonazol, itrakonazol, klotrimazol, amfoterisin B ve nistatin gradyan test şeritleri ile değerlendirilmiştir. 20 izolatın amfoterisin B'ye, 18 izolatın ise nistatin antifungaline karşı dirençli olduğu belirlenmiştir. İtrakonazolün 14 izolata ve klotrimazolün 3 izolata duyarlılığının doza bağımlı olduğu tespit edilmiştir. Sonuç olarak, azol grubu antifungaller ağırlıklı olarak oral maya enfeksiyonlarının tedavisinde kullanılabilir.

Anahtar Kelimeler

Antifungal direnç, Biyofilm, Candida, Gradiyent test

Proje Numarası

THD-2020-3352

Kaynakça

• Song, Y. B., Suh, M. K., Ha, G. Y., & Kim, H. (2015). Antifungal susceptibility testing with etest for Candida species isolated from patients with oral candidiasis. Annals of Dermatology, 27(6), 715. https://doi.org/10.5021/ad.2015.27.6.715
• Niimi, M., Firth, N. A., & Cannon, R. D. (2010). Antifungal drug resistance of oral fungi. Odontology, 98(1), 15-25. https://doi.org/10.1007/s10266-009-0118-3
• Whaley, S. G., Berkow, E. L., Rybak, J. M., Nishimoto, A. T., Barker, K. S., & Rogers, P. D. (2017). Azole antifungal resistance in Candida albicans and emerging non-albicans Candida species. Frontiers in Microbiology, 7, 2173. https://doi.org/10.3389/fmicb.2016.02173
• Kuriyama, T., Williams, D. W., Bagg, J., Coulter, W. A., Ready, D., & Lewis, M. A. O. (2005). In vitro susceptibility of oral Candida to seven antifungal agents. Oral Microbiology and Immunology, 20(6), 349-353. https://doi.org/10.1111/j.1399-302X.2005.00236.x
• Jiao, Y., Tay, F. R., Niu, L. N., & Chen, J. H. (2019). Advancing antimicrobial strategies for managing oral biofilm infections. International Journal of Oral Science, 11(3), 1-11. https://doi.org/10.1038/s41368-019-0062-1
• Dhale, R. P., Ghorpade, M. V., & Dharmadhikari, C. A. (2014). Comparison of various methods used to detect biofilm production of Candida species. Journal of Clinical and Diagnostic Research: JCDR, 8(11), DC18. https://doi.org/10.7860/JCDR/2014/10445.5147
• Khatri, S., Sumana, M. N., Mahale, R. P., & Kishore, A. (2015). Analysing three different screening methods for biofilm formation in clinical isolates of Candida. Journal of Evolution of Medical and Dental Sciences, 4(83), 14515-14524. https://doi.org/10.14260/jemds/2015/2065
• Sida, H., Shah, P., Pethani, J., Patel, L., & Shah, H. (2016). Study of biofilm formation as a virulence marker in Candida species isolated from various clinical specimens. International Journal of Medical Science and Public Health, 5(5), 842-6. http://journalsarchive.com/FILES/IJMSPH/04.%20Study%20of%20biofilm%20formation.pdf
• Shrestha, B. K., & Shakya, J. (2020). In-vitro evaluation of biofilm and hemolysis activity of candida albicans isolated from oral cavity. International Journal of Applied Sciences and Biotechnology, 8(4), 394-399. https://doi.org/10.3126/ijasbt.v8i4.32971
• Lewis, K. (2012). Persister cells: molecular mechanisms related to antibiotic tolerance. Anthony R.M. Coates (Ed), Antibiotic Resistance, (pp. 121-133). Springer
• Martins, C. H. G., Pires, R. H., Cunha, A. O., Pereira, C. A. M., de Lacorte Singulani, J., Abrão, F., Moraes, T. D., & Mendes-Giannini, M. J. S. (2016). Candida/Candida biofilms. First description of dual-species Candida albicans/C. rugosa biofilm. Fungal biology, 120(4), 530-537. https://doi.org/10.1016/j.funbio.2016.01.013
• Seneviratne, C. J., Wang, Y., Jin, L., Abiko, Y., & Samaranayake, L. P. (2010). Proteomics of drug resistance in Candida glabrata biofilms. Proteomics, 10(7), 1444-1454. https://doi.org/10.1002/pmic.200900611
• Tumbarello, M., Posteraro, B., Trecarichi, E. M., Fiori, B., Rossi, M., Porta, R., Donati, K. D. G., Sorda, M. L., Spanu, T., Fadda, G., Cauda, R., & Sanguinetti, M., (2007). Biofilm production by Candida species and inadequate antifungal therapy as predictors of mortality for patients with candidemia. Journal of Clinical Microbiology, 45(6), 1843-1850. https://doi.org/10.1128/jcm.00131-07
• Silva, S., Negri, M., Henriques, M., Oliveira, R., Williams, D. W., & Azeredo, J. (2011). Adherence and biofilm formation of non-Candida albicans Candida species. Trends in Microbiology, 19(5), 241-247. https://doi.org/10.1016/j.tim.2011.02.003
• Tumbarello, M., Fiori, B., Trecarichi, E. M., Posteraro, P., Losito, A. R., De Luca, A., Sanguinetti, M., Fadda, G., Cauda, R., & Posteraro, B. (2012). Risk factors and outcomes of candidemia caused by biofilm-forming isolates in a tertiary care hospital. PloS One, 7(3), e33705. https://doi.org/10.1371/journal.pone.0033705
• Kuramitsu, H. K., He, X., Lux, R., Anderson, M. H., & Shi, W. (2007). Interspecies interactions within oral microbial communities. Microbiology and Molecular Biology Reviews, 71(4), 653-670. https://doi.org/10.1128/MMBR.00024-07
• Thein, Z. M., Seneviratne, C. J., Samaranayake, Y. H., & Samaranayake, L. P. (2009). Community lifestyle of Candida in mixed biofilms: a mini review. Mycoses, 52(6), 467-475. https://doi.org/10.1111/j.1439-0507.2009.01719.x
• Kolenbrander, P. E., Palmer, R. J., Periasamy, S., & Jakubovics, N. S. (2010). Oral multispecies biofilm development and the key role of cell–cell distance. Nature Reviews Microbiology, 8(7), 471-480. https://doi.org/10.1038/nrmicro2381
• Flemming, H. C., & Wingender, J. (2010). The biofilm matrix. Nature Reviews Microbiology, 8(9), 623-633. https://doi.org/10.1038/nrmicro2415
• Gulati, M., & Nobile, C. J. (2016). Candida albicans biofilms: development, regulation, and molecular mechanisms. Microbes and İnfection, 18(5), 310-321. https://doi.org/10.1016/j.micinf.2016.01.00
• Miquel, S., Lagrafeuille, R., Souweine, B., & Forestier, C. (2016). Anti-biofilm activity as a health issue. Frontiers in Microbiology, 7, 592. https://doi.org/10.3389/fmicb.2016.00592
• Özcan Ateş, G. (2020). Ağızda Maya Yükünün Belirlenmesi ve İzole Edilen Candida Cinsi Mayaların Tanımlanması. (Tez no: 621102) [Yüksek Lisans Tezi, Çanakkale Onsekiz Mart Üniversitesi].
• Freeman, D. J., Falkiner, F. R., & Keane, C. T. (1989). New method for detecting slime production by coagulase-negative staphylococci. Journal of clinical pathology, 42(8), 872-874. http://dx.doi.org/10.1136/jcp.42.8.872
• Christensen, G. D., Simpson, W. A., Younger, J. J., Baddour, L. M., Barrett, F. F., Melton, D. M., & Beachey, E. H. (1985). Adherence of coagulase-negative staphylococci to plastic tissue culture plates: a quantitative model for the adherence of staphylococci to medical devices. Journal of Clinical Microbiology, 22(6), 996-1006. https://doi.org/10.1128/jcm.22.6.996-1006.1985
• Muadcheingka, T., & Tantivitayakul, P. (2015). Distribution of Candida albicans and non-albicans Candida species in oral candidiasis patients: Correlation between cell surface hydrophobicity and biofilm forming activities. Archives of Oral Biology, 60(6), 894-901. https://doi.org/10.1016/j.archoralbio.2015.03.002
• Kıvanç, M., & Er, S. (2020). Biofilm formation of Candida spp. isolated from the vagina and antibiofilm activities of lactic acid bacteria on the these Candida Isolates. African Health Sciences, 20(2), 641-648. https://doi.org/10.4314/ahs.v20i2.12
• Stepanović, S., Vuković, D., Hola, V., Bonaventura, G. D., Djukić, S., Ćirković, I., & Ruzicka, F. (2007). Quantification of biofilm in microtiter plates: overview of testing conditions and practical recommendations for assessment of biofilm production by staphylococci. Apmis, 115(8), 891-899. https://doi.org/10.1111/j.1600-0463.2007.apm_630.x
• National Committee for Clinical Laboratory Standards. (2004). Method for Antifungal Disk Diffusion Susceptibility Testing of Yeasts; Approved Guideline. (NCCLS Standard No: M44-A). https://clsi.org/standards/products/microbiology/documents/m44/
• National Committee for Clinical Laboratory Standards. (2002). Reference Method for Broth Dilution Antifungal Susceptibility Testing of Yeasts; Approved Standard. (NCCLS Standard No. M27-A2). https://clsi.org/standards/products/microbiology/documents/m44/
• Espinel-Ingroff, A., & Pfaller, M. A. (1995). Antifungal agents and susceptibility testing. Manual of clinical microbiology, 6th ed. American Society for Microbiology, Washington, DC, 1405-1414.
• Davey, K. G., Holmes, A. D., Johnson, E. M., Szekely, A., & Warnock, D. W. (1998). Comparative evaluation of FUNGITEST and broth microdilution methods for antifungal drug susceptibility testing of Candida species and Cryptococcus neoformans. Journal of Clinical Microbiology, 36(4), 926-930. https://doi.org/10.1128/JCM.36.4.926-930.1998
• Kronvall, G., & Karlsson, I. (2001). Fluconazole and Voriconazole Multidisk Testing of Candida Species for Disk Test Calibration and MIC Estimation. Journal of Clinical Microbiology, 39(4), 1422-1428. https://doi.org/10.1128/jcm.39.4.1422-1428.2001
• Blignaut, E., Messer, S., Hollis, R. J., & Pfaller, M. A. (2002). Antifungal susceptibility of South African oral yeast isolates from HIV/AIDS patients and healthy individuals. Diagnostic Microbiology and Infectious Disease, 44(2), 169-174. https://doi.org/10.1016/S0732-8893(02)00440-6
• Gogoi, M., Sharma, A., & Hazarika, N. (2015). Biofilm formation by bacterial isolates from patients on indwelling medical devices. Indian Journal of Medical Microbiology, 33(2), 319. https://doi.org/10.4103/0255-0857.154896
• Pathak, A. K., Sharma, S., & Shrivastva, P. (2012). Multi-species biofilm of Candida albicans and non-Candida albicans Candida species on acrylic substrate. Journal of Applied Oral Science, 20(1), 70-75. https://doi.org/10.1590/S1678-77572012000100013
• Mohandas, V., & Ballal, M. (2011). Distribution of Candida species in different clinical samples and their virulence: biofilm formation, proteinase and phospholipase production: a study on hospitalized patients in southern India. Journal of Global Infectious Diseases, 3(1), 4. https://doi.org/10.4103/0974-777X.77288
• Weerasekera, M. M., Wijesinghe, G. K., Jayarathna, T. A., Gunasekara, C. P., Fernando, N., Kottegoda, N., & Samaranayake, L. P. (2016). Culture media profoundly affect Candida albicans and Candida tropicalis growth, adhesion and biofilm development. Memorias do Instituto Oswaldo Cruz, 111(11), 697-702. https://doi.org/10.1590/0074-02760160294
• McCourtie, J., MacFarlane, T. W., & Samaranayake, L. P. (1985). Effect of chlorhexidine gluconate on the adherence of Candida species to denture acrylic. Journal of Medical Microbiology, 20(1), 97-104. https://doi.org/10.1099/00222615-20-1-97
• Samaranayake, L. P., MacFarlane, T. W., Lamey, P. J., & Ferguson, M. M. (1986). A comparison of oral rinse and imprint sampling techniques for the detection of yeast, coliform and Staphylococcus aureus carriage in the oral cavity. Journal of Oral Pathology & Medicine, 15(7), 386-388. https://doi.org/10.1111/j.1600-0714.1986.tb00646.x
• Kucharíková, S., Tournu, H., Lagrou, K., Van Dijck, P., & Bujdakova, H. (2011). Detailed comparison of Candida albicans and Candida glabrata biofilms under different conditions and their susceptibility to caspofungin and anidulafungin. Journal of Medical Microbiology, 60(9), 1261-1269. https://doi.org/10.1099/jmm.0.032037-0
• Konečná, K., Němečková, I., Diepoltová, A., Vejsová, M., & Janďourek, O. (2021). The Impact of Cultivation Media on the In Vitro Biofilm Biomass Production of Candida spp. Current Microbiology, 1-8. https://doi.org/10.1007/s00284-021-02452-6
• Dhanasekaran, D., Vinothini, K., Latha, S., Thajuddin, N., & Panneerselvam, A. (2014). Human dental biofilm: Screening, characterization, in vitro biofilm formation and antifungal resistance of Candida spp. The Saudi Journal for Dental Research, 5(1), 55-70. https://doi.org/10.1016/j.ksujds.2013.10.001
• Koga-Ito, C. Y., Lyon, J. P., & Resende, M. A. D. (2008). Comparison between E-test and CLSI broth microdilution method for antifungal susceptibility testing of Candida albicans oral isolates. Revista do Instituto de Medicina Tropical de São Paulo, 50(1), 7-10. https://doi.org/10.1590/S0036-46652008000100002
• Arendrup, M. C., Friberg, N., Mares, M., Kahlmeter, G., Meletiadis, J., Guinea, J., Kahlmeter, G., Andersen, C.T., Arikan-Akdagli, S., Barchiesi, F., Chryssanthou, E., Hamal, P., Järv, H., Klimko, N., Kurzai, O., Lagrou, K., Lass-Flörl, C., Matos, T., Muehlethaler, K., Rogers, T. R., Velegraki, A., & Arikan, S. (2020). How to interpret MICs of antifungal compounds according to the revised clinical breakpoints v. 10.0 European committee on antimicrobial susceptibility testing (EUCAST). Clinical Microbiology And Infection, 26(11), 1464-1472. https://doi.org/10.1016/j.cmi.2020.06.007