Floresanlı Yerinde Hibritleme ve Geleneksel Kültür Yöntemlerini Kullanılarak Soğutma Kulesi Örneklerinde Fungusların Tespiti

Yıl 2017, Cilt: 8 Sayı: 1, 68 - 75, 28.04.2017

Duygu Kadaifçiler Zuhal Zeybek

Öz

Doğada ve insan yapımı
su sistemlerinde yaygın olarak bulunan Eurotiales üyesi fungusların varlığının tespiti, fırsatçı enfeksiyon ve
alerjilere yol açabilen bu funguslara karşı alınabilecek önlemler açısından
önemlidir. Bu çalışmada soğutma kulesi örneklerindeki fungusların varlığı, hem
floresanlı yerinde hibritleme (FISH) hem de kültür yöntemiyle araştırılmış, her
iki yöntemin sonuçları karşılaştırılmıştır. Bu amaçla İstanbul’daki farklı
soğutma kulelerine ait toplam 40 adet su ve biyofilm örnekleri alınarak
incelenmiştir. Kültür yönteminde tüm örnekler, ön işlemlerden sonra izolasyon
besiyerine ekilmiştir. Ekimlerin sonuçları 14 gün sonunda değerlendirilmiştir.
FISH yönteminde ise; tüm örnekler fikse edilip uygun problar kullanılarak
hibridizasyonu yapıldıktan sonra epifloresan mikroskopta incelenmiştir. FISH
yöntemi ile 3 gün gibi kısa bir sürede sonuç elde edilmiştir. Ayrıca bu yöntem ile incelenen örneklerin tamamında, kültür yönteminde ise incelenen örneklerin
%97.5’in de fungus tespit edilmiştir. Kültür yöntemi ile en çok izole edilen
fungusların Aspergillus ve Penicillium cinsine ait oldukları
belirlenmiştir. Çalışma sonucunda her iki yöntemin beraber kullanılması
önerilmektedir.

Anahtar Kelimeler

Aspergillus, kültür bağımsız yöntem, Eurotiales, floresanlı yerinde hibritleme

Detection of Fungi in Cooling Tower Samples Using Fluorescent In Situ Hybridization and Traditional Culture-Dependent Methods

Öz

The
determination of the presence of Eurotiales members, which are widely
distributed in nature and in man-made aquatic systems, is important for taking
measures against those capable of creating opportunistic infections and
allergies. In this study, the presence of fungi in cooling-tower samples was determined
by using two different methods, one being fluorescent in situ hybridization
(FISH), the second culturing; both sets of results were compared with each
other. For this purpose, a total of 40 water and biofilm samples were taken
from different cooling towers in Istanbul for investigation. In the culture
method, all samples were applied to the isolation medium after pretreatment.
The results of the applications were evaluated after 14 days. With the FISH
method, all samples were fixed and hybridized by using suitable probes and were
investigated under an epifluorescent microscope. Results were obtained after a
short period of three days. The FISH method was able to detect 100% of fungi in
all samples, whereas the culture method was able to detect 97.5% of the fungi
in the samples. With culture method, the most isolated fungi were from the Aspergillus and Penicillium genera. As a result of the study, both methods are
recommended to be used in conjunction.

Anahtar Kelimeler

Aspergillus, culture-independent method, Eurotiales, fluorescent in situ hybridization

Kaynakça

• Amann, R.I., Fuchs, B., Behrens, S., The Identification of Microorganisms by Fluorescence İn Situ Hybridisation, Current opnion in Biotechnology, 12(3): 231–236 (2001).
• Amann, R.I., Ludwig, W., Schleifer. K.H., Phylogenetic İdentification and İn Situ Detection of Individual Microbial Cells without Cultivation. Microbiological Reviews. 59: 143-169 (1995).
• Arvanitidou, M., Kanellou, K., Constantinides, T.C., Katsouyannopoulos, V., The Occurrence of Fungi in Hospital and Community Potable Waters, Letters in Applied Microbiology, 29: 81-84 (1999).
• Asan, A., Ekmekçi, S., Contribution to the Colonical and Morphological Characteristics of Some Aspergillus Species Isolated from Soil, Ege University Journal of The Faculty of Science, 25(1): 121-139 (2002).
• Asan, A., Kırgız, T., Şen, B., Çamur-Elipek, B., Güner, U., Güher, H. :Isolation, Identification and seasonal distribution of airborne and waterborne fungi in Terkos lake (İstanbul-Turkey), Journal of Basic Microbiology, 43(2): 83-95 (2003).
• Asan, A., İlhan, S., Şen, B., Potoğlu Erkara, İ., Filik, C., Çabuk, A., Demirel, R., Türe, M., Sarıca Ökten, S., Tokur, S.,: Airborne Fungi and Actinomycetes Concentrations in the Air of Eskişehir City (Turkey), Indoor and Built Environment, 13: 63-74 (2004).
• Baker, B.J., Lutz, M.A., Dawson, S.C., Bond, P.L., Banfield, J.F., Metabolically Active Eukaryotic Communities in Extremely Acidic Mine Drainage, 70(10): 6264-6271 (2004).
• Baschien, C., Manz, W., Neu,T.R., Marvanova´, L., Szewzyk, U., In Situ Detection of Freshwater Fungi in an Alpine Stream by New Taxon-Specific Fluorescence In Situ Hybridization Probes, Applied and Cooling tower Microbiology, 74(20): 6427-6436 (2008).
• Choudhary, S.G., Emerging Microbial Control Issues in Cooling Water Systems. Hydrocarbon Processing, 77(5): 91-102 (1998).
• Demirel, R., Ilhan, S., Asan, A., Kınacı, E., Oner, S., Microfungi in Cultivated Fields in Eskişehir Provience (Turkey), Journal of Basic Microbiology, 45(4): 279-293 (2005).
• Domingos Arantes, T., Cordeiro Theodoro R., de Melo Teixeira, M., Bagagli, E., Use of Fluorescent Oligonucleotide Probes for Differentiation between Paracoccidioides brasiliensis and Paracoccidioides lutzii in Yeast and Mycelial Phase, The Memórias do Instituto Oswaldo Cruz, Rio de Janeiro, 112(2): 140-145 (2017).
• Ellis, M.B., Dematiaceous Hyphomycetes, The Eastern Press, London, UK, (1971).
• Gagnon, G.A., Slawson, R,M., An Efficient Biofilm Removal Method for Bacterial Cells Exposed to Drinking Water, Journal of Microbiological Methods, 34(3): 203- 214 (1999).
• Göksay Kadaifciler, D., Demirel, R., Fungal Biodiversity and Mycotoxigenic Fungi in Cooling-Tower Water Systems in Istanbul, Turkey, DOI: 10.2166/wh.2017.274 (2017).
• Guarro, J., Gene, J., Stchigel, A., Developments in Fungal Taxonomy, Clinical Microbiology Reviews, 12(3): 454-500 (1999).
• H.L. Barnett, B.B. Hunter, Illustrated genera of imperfect fungi, APS Press, St. Paul-Minnesota, USA, 1999.
• Ilhan, S., Demirel, R., Asan, A., Bayçu, G., Kınacı, E., Colonial and Morphological Characteristics of Some Microfungal Species Isolated from Agricultural Soils in Eskişehir Province (Turkey), 30: 95-104 (2006).
• Jobard, M., Rasconi, S., Sime-Ngando, T., Fluorescence in Situ Hybridization of Uncultured Zoosporic Fungi: Testing with Clone-FISH and Application to Freshwater Samples using CARD-FISH, Journal of Microbiological Methods, 83: 236-243 (2010).
• Kantarcıoğlu, A. S., Yücel, A., Aspergillus Cinsi Mantarlar ve İnvaziv Aspergilloz: Mikoloji, Patogenez, Laboratuvar Tanımı, Antifungallere Direnç ve Duyarlılık Deneyleri, Cerrahpaşa Tıp Dergisi, 34(3): 140-145 (2003).
• Kasapgil İnce, B., Usenti, I., Eyigor, A., Ayman Oz, N., Kolukirik, M., Ince, O., Analysis of Methanogenic Archaeal and Sulphate Reducing Bacterial Populations in Deep Sediments of the Black Sea, Geomicrobiology Journal, 23: 285–292 (2006).
• Klich, M.A., Identification of common Aspergillus species, Centraalbureau voor Schimmelcultures, Utrecht, Netherlands, (2002).
• Li, S., Spear, R.N., Andrews, J.H., Quantitative Fluorescence in Situ Hybridization of Aureobasidium pullulans on Microscope Slides and Leaf Surfaces, Applied and Cooling tower Microbiology, 63(8): 3261-3267 (1997).
• Lischewsk, A., Amann, R.I., Harmsen, D., Merkert, H., Hacker, J., Morschhauser, J., Specific Detection of Candida albicans and Candida tropicalis by Fluorescent İn Situ Hybridization with an 18S rRNA-Targeted Oligonucleotide Probe, Microbiology, 142: 2731-2740 (1996).
• Moter, A., Göbel, U.B., Fluorescence in Situ Hybridization (FISH) for Direct Visualization of Microorganisms, Journal of Microbiological Methods, 41: 85-112 (2000).
• Öner, M., Mikoloji I, Myxomycetes, Phycomycetes, Ascomycetes, 4. Baskı. Ege Üniversitesi Fen Fakültesi Kitaplar Serisi no 53. Ege Üniversitesi Basımevi, Bornova, İzmir. (1998).
• Sime, A.D., Abbott, L.L., Abbott, S.P., Mounting Medium for Use in İndoor Air Quality Spore-Trap Analyses, Mycologia., 94: 1087-1088 (2002).
• Spear, R.N., Li, S., Nordheim, E.V., Andrews, J.H., Quantitative İmaging and Statistical Analysis of Fluorescence Hybridization (FISH) of Aureobasidium pullulans, Journal of Microbiological Methods 35: 101-110 (1999).
• Szymanska, J., Evaluation of Mycological Contamination of Dental Unit Waterlines, Annals of Agricultural and Cooling tower Medicine,12:153-155 (2005).
• Yazıcıoğlu, M., Asan, A., Ones, U., Vatansever, U., Şen, B., Ture, M., Bostancıoğlu, M., Pala, O.: Indoor Airborne Fungal Spores and Home Characteristics in Asthmatic Children from Edirne Region of Turkey, Allergologia et Immunopathologia, 32(4): 197-203 (2004).