Aspergillus Fumigatus Identification and Molecular Character

Aspergillus Fumigatus Identification and Molecular Character IDENTIFICATION AND MOLECULAR CHARACTERIZATION OF Aspergillus fumigatus FROM SOIL R. V. Shalini, and Dr. K. Amutha ABSTRACT: Soil was collected, serially diluted and pure culture obtained; slant was prepared in potato dextrose agar and maintained throughout the study. Morphological, microscopical and macroscopically identification were carried out on the isolated organism. DNA was isolated from the 24 hour culture, for ITS-PCR amplification. DNA was amplified by mixing the template DNA (50nm) with the polymerase reaction buffer, dNTP mix, primers and Taq. Polymerase chain reaction (PCR) was performed in a total volume of 50Â µL reaction mixture. The PCR product was mixed with loading buffer (8Â µL) containing 0.025% bromophenol blue, 40% w/v sucrose in water and then loaded in 2% agarose gel with 0.1% of ethidium bromide and the amplified product was visualized under a UV trans illuminator for further examination. The PCR products were finally sequenced using the help of an automated DNA sequencer at progen Ltd (Salem, India) and analyzed with the BLAST program provided by the National Center f or Bio-technology information (NCBI) to confirm the fungal species. The current study demonstrates that DNA genome containing 18S rRNA has a high degree of analytical sensitivity and specificity (100%) for the detection of a wide range of fungi. OBJECTIVE: To isolate, identify and characterize Aspergillus fumigatus using molecular biological methods. MATERIALS AND METHODS: The soil was collected from different places, pooled together allowed to be dried at room temperature. The morphology based identification of Aspergillus was done which includes the size, shape, colour, ornamentation of spore and mode of attachment. Unfortunately a lot of difficulties arose for phenotypical identification of this fungus due to its unstable characteristics. Comparatively a DNA sequence-based identification format appeared to be the most promising in terms of its speed, ease, objectivity and reliability for species identification. RESULTS: The preliminary morphology based studies showed the isolated fungi as a species of aspergillus.However after the DNA isolation followed by sequencing it was concluded that the particular species identified as Aspergillus was Aspergillus fumigatus. KEY WORDS: Aspergillus, serial dilution, DNA, Sequenced. INTRODUCTION: The presence of organic matter in the soil affects the quantity and quality of microbes in the soil. The development of micro fungi in the soil is favoured by soils having acidic reaction and aerobic condition which is likely present in the soil. However the amount of degradation in the soil is brought about by the organisms present in the soil. 1The rate at which the organic matter is decomposed is inter related with soil microbes. (Arunachalam et al., 1997). Microorganisms come in various sizes and shapes and is determined by the soil ph., temperature, available moisture, degree of aeration, availability of nutrients in the soil etc. The genus of spore forming fungi is found worldwide out of which Aspergillus is the most dominant species and is ubiquitoes.Out of that 95% is occupied by Aspergillus fumigatus. The other pathogenic forms of Aspergillus species are Aspergillus flavus, Aspergillus niger, Aspergillus nidulans, Aspergillus terreus etc. This fungi exists only in mycelial f orm, and is thermo tolerant capable of growing at temperatures between 15-53Â °c.Being a spore producing fungi the spores gets dispersed by wind in the atmosphere. 2Aspergillus fumigatus is the most common among all the airborne saprophytic fungal pathogens in immune compromised patients mostly in developed countries (Latge, 1999). It is the main pathogenic agent of various diseases caused in humans including invasive pulmonary aspergillosis, aspergilloma and allergic bronchopulmonary aspergillosis (Tomee van der Werf, 2001) – the former is a frequent cause death in immune compromised patients. The possession of different virulent traits gives A. fumigatus the ability to cause these diseases. It is a known fact that other members of the genus Aspergillus are either less pathogenic or non- pathogenic. Identification of the most common and important species remains problematic due to the variability in the phenotypic characters. However a validated and a careful approach of phenotypic classification (taxonomy) together with phylogenetic treatment of DNA sequence data is a prerequisite for a reliable and a rapid identification. In our inve stigation we used the molecular techniques (sequencing) for the reliable identification rather the identification based on their microscopic and few physiological features. MATERIALS METHODS: Collection of soil samples: Soil samples were collected from different places (in and around Chengalpattu). The surface deposits were removed to a depth of about 10 cm and the exposed soil was collected to a depth of 2-3cm. The collected soil samples were stored in zip locked covers stored in refrigerator temperature for further analysis. The collected soil samples were passed through a sieve to remove the stones and other impurities. Isolation of fungi: The glass wares were sterilized in an autoclave to a temperature of 120Â °c for twenty minutes. The chemicals were of analytical grade (Himedia). The method used for the isolation of fungi from soil was serial dilution method. 1 gm of soil was weighed and mixed in 10ml of double distilled sterile water. This was used for preparing serial dilutions. 1 ml of the final dilution (10-6 ) was pipetted into the prepared potato dextrose agar media (PDA) amended with a suitable antibiotic Chloramphenicol (12mg/100ml). The plates were incubated at 30Â °c for about seven days. Fungi that appeared on petriplates were isolated. The isolates were picked up based on apparent dissimilarity of cultural characteristics and purified. The purified isolates were identified according to the genera on the basis of cultural characteristics such as nature of growth, spore colour, and pigment production, and on morphological characteristics of mycelia and fruiting bodies (Domsch etal., 1980; Raper and Fenne ll 1965) and maintained in agar slants for future use3. Isolation of DNA: Genomic DNA was extracted from 24 hour old culture. Measured 100 micro gram of mycelium into a sterile 1.5- micro centrifuge tube. Simultaneously ground 1 microgram of dried (vacuum filter mycelium first) in a mortar and pestle treated with liquid nitrogen 5-6 times. Poured the frozen powder into the Eppendorf tube. Added 660 750 Â µl of lysis buffer and 10 Â µl of B-mercaptor.Vortexed the mixture for a few seconds. And Incubated at 65Â °C for 1 hour. Used a water bath for incubation. Centrifuged at a speed of 3400 rpm for 5 minutes at room temperature and aspirated out the top layer.Transfered the top aqueous layer into a fresh Eppendorf tube discarded the bottom layer. Measured out 700 Â µl of chloroform, isoamyl alcohol (24:1) into Eppendorf tube and adjusted the volume to meet a 1:1 ratio of aqueous phase.Vortexed the mixture for a few seconds. Centrifuged at a speed of 12000 rpm for 10 minutes at room temperature and aspirated out 550 600 Â µl of the top layer. Transfered the top aqueous layer into Eppendorf tube and discarded the bottom layer. Added 0.1volume of 3m potassium acetate and 0.7 volume of isopropanol. Mixed well by inverting the tube not by vortexing.Centrifuged for about 10 minutes and discarded the supernatant. Added 0.5 mL of ice cold ethanol (70% and inverted the tube gently, again it was centrifuged for about 5 minutes in a spinner) finally the pellets were resuspended in 100Â µl of TE buffer (PH-8). After further purification DNA was quantified spectrophotpmetrically and the quality was analyzed in 0.9% agorose gel. Amplification of 18srRNA by PCR: For ITS-PCR amplification, DNA was amplified by mixing the template DNA (50nm) with the polymerase reaction buffer, dNTP mix, primers and Taq polymerase chain reaction (PCR) was performed in a total volume of 50Â µL reaction mixture containing Primer (2Â µM/Â µL) 8.0Â µL 10X Buffer 5.0 Â µL 2mM dNTP Mix 5.0Â µL Taq DNA polymerase (5U/Â µL) 0.5Â µL Template DNA (50ng) 2.0Â µL Sterile distilled water 29.5Â µL Total volume 50.0Â µL PCR amplification condition: Amplification was carried out in a primus advanced gradient thermocycler. The PCR was programmed with an initial denaturing at 94Â °C for 5 min, followed by 30 cycles of denaturation at 94Â °c for 30 seconds, annealing at 61Â °c for 30 seconds, and extension at 70Â °c for 2 minutes and a final extension at 72Â °c for 7 minutes. The PCR product was mixed with loading buffer (8Â µL) containing 0.025% bromophenol blue, 40% w/v sucrose in water and then loaded in 2% agarose gel with 0.1% of ethidium bromide and the amplified product was visualized under a UV trans illuminator for further examination. (Sequencing) Sequencing of ITS region for identification of isolated fungi : Chosen Samples of the genomic DNA containing 18S rRNA were shortlisted for more specific species confirmation by using DNA sequencing. The sequenced PCR product was aligned with other isolate sequences from NCBI genbank for identification. The PCR products were finally sequenced using the help of an automated DNA sequencer at progen Ltd (Salem, India) and analyzed with the BLAST program provided by the National Center for Bio-technology information (NCBI) to confirm the fungal species. RESULTS: Macroscopic and Microscopic Analysis: Analysis of the isolated Aspergillus species showed variation in the colony colours, texture, and reverse side colours (table 1 and 2). The morphological microscopic and molecular characteristics showed that the isolate is Aspergillus fumigatus (details given in table 1and 2). Morphological characters of colony (table1) Characteristics Aspergillus fumigatus Surface colour Margins Reverse side Growth Green to dark green Entire Yellow Rapid Microscopic characteristics (table2) Characteristics Aspergillus fumigatus Hyphae Branched septate Conidiophore Present Vesicle Dome shaped Conidia Present Phialides Uniseriate Fruiting body Cleistothecia Fig A1 Fig A2 Morphological characterization of Aspergillus species on potato dextrose agar A1-Aspergillus fumigatus surface colour, A2-Reverse side of the colony. DNA sequencing of ITS region for identification of species: The species of fungi from the PCR sample was identified by DNA sequencing of the internally transcribed spacer (ITS) region of rRNA gene. Segments of the entire ITS regions, including partial 5.8S rRNA and internal transcribed spacer 2, complete sequence, 28S rRNA,partial sequence were amplified using the primer PGF04 5’-GGC ATC GGC C-3’. Amplification of the ITS region of strain Aspergillus fumigatus had a size of 1703bp. It was submitted to the NCBI and the accession number KC 119199 was received. M 1 2 Fig A3 represent the banding pattern of Aspergillus fumigatus from PCR reactions Lane M= Marker, Lane 1= Aspergillus fumigatus, lane 2=Aspergillus fumigatus. DISCUSSION: Detection of A. fumigatus is of great concern because it is a dangerous allergen associated with aspergillosis 5(Abraca et al., 1994; Schuster et al., 2002; Noonimabcet al.2009; Edwin et al., 2010; Gautam et al., 2011). This highlights the importance of correct identification and taxonomical differentiation between different species of Aspergillus. The taxonomy of Aspergillus has always been complex due to its great number of species (nearly 250), which have very few differences. The identification of different Aspergillus species, on the basis of their morphological characters (example, colony colours, and reverse side) is one of the oldest and most adopted methods. Some of the species of Aspergillus have the same morphological features which make it difficult to distinguish between them it is also a time consuming process and may not be accurate (Klich and Pitt, 1988; Samson et al., 2004)6. This shows that morphological and microscopical characters are not enough for fungal identification and it renders the need of molecular techniques for correct species identification. Molecular characterization on the other hand, is a rapid and a quick procedure which requires minimal handling of pathogens. It also helps in distinguishing morphologically, similar fungal species. Several similar studies on the application of PCR technology were used for the identification and detection of fungi, by using internal transcribed spacer (ITS) were already been studied and published7 by several scientist (Henson and French, 1993; Marek et al., 2003; Haughland et al., 2004; Druzhinina etal., 2005).. Many more such studies were also carried out very recently by God et and Munaut (2010) in the differentiation of Aspergillus flavus, A.parasiticus, A.tamarii and A.nomius by PCR-RAPD markers. Similarly, Leema et al. (2010) confirmed the species A. flavus by verifying; using the molecular methods that is, by amplification of the internally transcribed spacer regions. By using the help of RAPD-PCR, 8Khan et al. (2007) studied diversity in various Aspergillus niger isolates sourc ed from pigeon pea fields .Several molecular techniques have been tested to classify different Aspergillus species like random amplification of polymorphic DNA (RAPD) (Yuan et al., 1995), the internal transcribed spacer (ITS) region (Kumeda and Asao, 1996; Henry et al., 2000; Kumeda and Asao, 2001; Rigo et al., 2002) and the aflatoxin gene cluster (Chang et al., 1995; Watson et al., 1999; Tominaga et al., 2006). In this study care was taken to choose the genomic DNA containing 18sRNA specifics primers that were helpful in amplifying medically important fungi. The genomic DNA containing 18s rRNA was the right candidate for detection of fungus as it is a mutli-copy gene which evolves slowly and is conserved among fungi. The present study proves that the genomic DNA containing 18s rRNA based PCR is suitable for probing large range of medically significant fungi owing to its higher level of analytical sensitivity and specificity. CONCLUSION: In this present study we had shown that molecular techniques are rapid and best for identification of fungi than the traditional morphological methods for early diagnosis and treatment of fungal infections. The goal of our study was to identify a practical, quick, cheap, method for the identification of A. fumigatus, the most common of the Aspergillus pathogens. REFERENCES: Arunachalam K, Arunachalam A, Tripathi RS, Pandey HN. 1997 – Dynamics of microbial population during the gradation phase of a selectively logged subtropical humid forest in north east India. Tropical Ecology 38, 333–341. Sirida Youngchim,1,2 Rachael Morris-Jones,1 Roderick J. Hay3 and Andrew J. Hamilton1 Production of melanin by Aspergillus fumigatus Journal of Medical Microbiology 2004, 53, 175–181. Domsch, K.H.,Gams W. and Anderson T.H. 1980.Compendium of soil fungi, vol 1.IHW-Verlag,Eching.,Raper,K.B.andFennel,D.I.1965.The genus Aspergillus (Baltimore: Williams Wilkins). Ferrer C, Colom F, Frases S, Mulet E, Abad JL, Alio JL:Detection and identification of fungal pathogens by PCR and by ITS2 and 5.8S ribosomal DNA in eye infections. J ClinMicrobiol 2001, 39(8): 2873-2879. Abraca ML, Bragulat G, Cabanes FJ 1994. Ochratoxin A production by strains of Aspergillus flavus var. niger . Appl. Environ. Microbiol. 60:2650-2652. Klich MA, Pitt JI 1988. Differentiation of Aspergillus flavus from Aspergillus parasiticus and other closely related species.Trans. Br.Mycol. Soc.91:99-108. Henson J, French R 1993. The polymerase chain reaction and plant disease diagnosis. Ann. Rev. Phytopathol.31:81-109. Khan MR, Anwer MA, Mohiddin FA 2007. Molecular diversity in Aspergillus isolates collected from pigeonpea field in Aligarh region.Environ. Biol. Conserv. 12:59-64. Godet F, Munaut F 2010. Molecular strategy for identification in Aspergillus section flavi. FEMS Microbiol. Lett. 304:157-168. Leema G, Kaliamurthy J, Geraldine J, Thomas PA 2010. Keratitis due to Aspergillus flavus: Clinical profile, molecular identification of fungal strains and detection of Aflotoxin production.Mol. Vision 6:843-854. Yuan G, Liu C, Chen C 1995. Differentiation of Aspergillus parasiticus from Aspergillus sojae by Random Amplification of Polymorphic DNA. Appl. Environ. Microbiol. 61:2384-2387. Kumeda Y, Asao T 2001. Heteroduplex panel analysis a novel method for genetic identification of Aspergillus section flavi strains. Appl. Environ. Microbiol. 67:4084-4090. Chang PK, Ehrlich KC, Bhatnagar JD, Cleveland TE 1995. Increased expression of Aspergillus parasiticus aflR, encoding a sequence specific DNA-binding protein, relieves nitrate inhibition of aflatoxin biosynthesis. Appl. Environ. Microbiol. 61:2372-2377.