Research Article Isolation and Molecular Identification of … · 2019. 7. 31. ·...

Hindawi Publishing CorporationBioMed Research InternationalVolume 2013 Article ID 619576 5 pageshttpdxdoiorg1011552013619576

Research ArticleIsolation and Molecular Identification of Keratinophilic Fungifrom Public Parks Soil in Shiraz Iran

Keyvan Pakshir Moosa Rahimi Ghiasi Kamiar Zomorodian and Ali Reza Gharavi

Department of Parasitology and Mycology Basic Sciences in Infectious Diseases Research Center School of MedicineShiraz University of Medical Sciences Shiraz 71345-45794 Iran

Correspondence should be addressed to Keyvan Pakshir pakshirkgmailcom

Received 30 April 2013 Accepted 27 June 2013

Academic Editor Abdelwahab Omri

Copyright copy 2013 Keyvan Pakshir et alThis is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Introduction Keratinophilic fungi are an important group of fungi that live in soil The aim of this study was to isolate andidentify keratinophilic fungi from the soil of different parks in Shiraz Materials and Methods A total of 196 soil samples from43 parks were collected Isolation of the fungi was performed by hair bait technique The isolated colonies were identified bymorphologic feature of macro- and microconidia and molecular method using DNA sequence analysis ITS region of ribosomalDNA was amplified and the PCR products were sequenced Results 411 isolates from 22 genera were identified Fusarium (238)Chrysosporium (1313)Acremonium (1265) Penicillium (1239)Microsporum gypseum (194) Bionectria ochroleuca (121)Bipolaris spicifera (121) Scedosporium apiospermum (082) Phialophora reptans (082) Cephalosporium curtipes (049)Scedosporium dehoogii (024) Ochroconis constricta (024) Nectria mauritiicola (049) Chaetomium (049) Scopulariopsis(024) Malbranchea (024) and Tritirachium (024) were the most important isolates Most of the fungi were isolated fromthe soils with the PH range of 7 to 8 Conclusion Our study results showed that many keratinophilic fungi isolated from the parkssoil are important for public health and children are an important group at a high risk of being exposed to these fungi

1 Introduction

Keratinophilic fungi are ecologically an important group offungi which could be found in soil [1] Some groups of thesefungi are causative agents of cutaneous fungal infectionsnamed dermatophytosis and the other saprophyte fungimainly represent hyalohyphomycosis [2 3]Theprevalence ofthese fungi depends on different factors such as the presenceof creatinine in the soil pH and geographical location [1]Some of these fungi such as dermatophytes are well knownto cause tinea infections which could be transmitted fromsoil to humans In general soil could be considered as areservoir for human infection Forests farmyards park soilsand sediments of the rivers and oceans containing humusand organic materials are the best candidates for growth ofkeratinolytic and saprophytic fungi [4]

During the past years many researchers reported aboutisolation of keratinophilic fungi around the world [5ndash14]Also a lot of reports were available about the isolation ofgeophilic dermatophytes and keratinophilic fungi from thesoils of many parts of Iran and also dermatophytosis due to

the geophilic fungi during the last decade [15ndash18] Nowadaysmost people spend their time with their children in the parksfor fun and are potentially at risk for direct contact with soiland being exposed to keratinophilic fungi [4] Shiraz is oneof the most populous cities of Iran and the capital of Farsprovince which is located in the southwest of Iran It has amoderate towarmclimate (minus3 to 40∘C)with rain falling about350mmyear land area of approximately 225 km2 and morethan one million population Up to now there were no dataabout keratinophilic fungi in soil of Shiraz and no reportsabout the molecular identification of these fungi aroundIran Thus the present study aimed to isolate and identifykeratinophilic fungi from soil of the popular parks in Shirazby molecular and morphological analysis

2 Materials and Methods

21 Sample Collection In this descriptive study 196 soilsamples were collected from various sites of 43 different parksaround Shiraz during spring 2011The samples were collected

2 BioMed Research International

from the superficial layer of the soil whose depth did notexceed 5ndash10 cm by using an iron spatula In doing so 300ndash400 gramof soil was collected in sterile polyethylene bags andbrought to laboratory for further processing

22 Measuring of Soil pH PH of each soil sample was meas-ured after preparation of soil suspension (one gram of soil tofive mL deionized water) using pH meter [17 19]

23 Isolation and Identification of the Isolates We used Van-breuseghemrsquos hair bait technique for isolation of keratino-philic fungi [20] Briefly each soil sample was thoroughlymixed and about 70 gram of the soil was packed in a sterile90mm Petri dish Then several pieces of sterile healthychildren hair fragments were dispersed over the surfaceof the soil samples and moistened with sterile distilledwater supplemented with antibiotic solutions chlorampheni-col (02 gmL) and strepto-penicillin (1000 IUmL) All thebaited soil Petri dishes were incubated at room temperature(20ndash25∘C) in the dark for 2 months and got moistened ifnecessary After observation of colony growth around thehairs the colonies were subcultured on Sabouraudrsquos dextroseagar (SDA)with andwithout chloramphenicol (50mgL) andcycloheximide (500mgL) and purifiedThe fungi were iden-tified based on the conventionalmethod (colonymorphologyand macro- and microconidia characteristics) and DNA-based identification techniques

24 DNA-Based Identification Techniques (DNA SequenceAnalysis) Molecular identification of the unknown isolateswas achieved by DNA sequence analysis First the fungi weregrown in flasks containing Sabouraudrsquos dextrose broth andincubated at 25∘C for several days using shaker rotator Aftercolony growth the culture was filtered (Millipore USA) andthe fungal mass was washed with distilled water for severaltimes and stored in a freezer (minus20∘C) for further processing

DNAwas extracted by using Lee technique [21] withmildmodification First the frozen mycelium mass was smashedby mechanical pressure using sterile pounder and liquidnitrogen The acquired powder was then mixed with lysesbuffer and the DNA was extracted The ITS1ndash58SndashITS 2rDNA was amplified using ITS1 and ITS4 as forward andreverse primers as described by White et al [22] Amplifi-cation was performed in 50 120583L reaction volumes containing5 120583L of 10times buffer MgCl

2(25mm) 15 120583L dNTP (10mM)

05 120583L 05 120583L of each 02Mm primer (ITS1 31015840-TCC-GTA-GGT-GAA-CCT-GCG-G-51015840 and ITS4 31015840-TCC-TCC-GCT-TAT-TGA-TAT-GC-51015840) TaqPolymerase (125U) 05 120583LDNAsample 1 120583L and distilled water 405 120583L The PCR reactionwas carried out using a Thermal Cycler (R corbet cg1-96)with the following conditions denaturation at 94∘C for 5min34 cycles of (30 s at 94∘C 45 s at 56∘C and 45 s at 72∘C)extension at 72∘C for 7min and storage at 4∘C Negativecontrols were also used in each set of reactions The finalproducts were analyzed by electrophoresis on 12 agarosegel (Sigma) and stained with 05120583gmLminus1 ethidium bromideIn addition the PCR products were sent for sequencingin both directions (Bioneer Korea) The sequence results

Figure 1 Agarose gel electrophoresis and PCRproducts bands lines1 and 2 (marker) lines 3ndash12 (up) and 2ndash8 (down) are PCR productsof many unknown fungi

were processed by using the web-based blasting programbasic local alignment search tool (BLAST) at the NCBI site(httpwwwncbinlmnihgovBLAST) and the data werecompared with the NCBIGenebank database [23]

25 Data Analysis The study data were analyzed throughFisher exact test and Chi-square test

3 Results

PCR products bands on gel agarose are presented in Figure 1From the 196 soil samples a total of 411 colonies of ker-atinophilic fungi were isolated from 43 parks The fun-gal isolates belonged to 22 genera as follows Fusarium(2530) Penicillium (1313)Chrysosporium (1313)Acre-monium (1265) Aspergillus (1192) Mucor (948) Pae-cilomyces (413) Microsporum (242) Bipolaris (145)Bionectria (121) Pseudallescheria (073) Phialophora(073) Alternaria (073) Nectria (048) Cephalospo-rium (048) Chaetomium (048) Scopulariopsis (024)Scedosporium (024) Verticillium (024) Malbranchea(024)Tritirachium (024) andOchroconis (024)Moredetails about the species are presented in Table 1

Fusarium spp was the most common fungal isolateamong the parks Besides eight species of Microsporumgypseum were isolated from four parks with soil pH betweenseven and nine and two species ofMicrosporum fulvum fromone park with the same pH Most of the fungi were isolatedfrom the soil samples with pH between 7 and 8 (6642) andno colony growthwas seen in pH gt 9 as shown in Table 2Thestudy results revealed no significant correlation between soilpH and fungal speciesMore details about the isolates and soilpH are presented in Table 2

4 Discussion

The keratinolytic activity of keratinophilic fungi is importantfor ecology and has attracted many researchersrsquo attentionaround the world Keratinophilic fungi play an importantrole in the natural degradation of keratinized residues in thesoil [24 25] Some types of these fungi such as geophilicdermatophytes live in soil and could be transmitted to

BioMed Research International 3

Table 1 Frequency of keratinophilic fungi isolated from soils withdifferent PH

Fungal genuspH

6-7 7-8 8-9 gt9119899 119899 119899 119899

Aspergillus 0 0 31 1135 18 1343 0 0Acremonium 0 0 35 1282 17 1268 0 0Alternaria 0 0 2 073 1 075 0 0Scopulariopsis 0 0 1 036 0 0 0 0Ochroconis 0 0 1 036 0 0 0 0Bipolaris 0 0 4 146 2 149 0 0Bionectria 0 0 4 146 1 075 0 0Cephalosporium 0 0 1 036 1 075 0 0Paecilomyces 2 50 7 256 8 597 0 0Scedosporium 0 0 4 145 0 0 0 0Tritirachium 0 0 1 036 0 0 0 0Penicillium 2 50 36 131 16 1194 0 0Fusarium 0 0 66 241 38 2835 0 0Phialophora 0 0 2 073 1 075 0 0Chrysosporium 0 0 39 1428 15 1119 0 0Chaetomium 0 0 2 073 0 0 0 0Mucor 0 0 22 842 16 1194 0 0Microsporum 0 0 8 366 0 0 0 0Malbranchea 0 0 1 036 0 0 0 0Nectria 0 0 2 073 0 0 0 0Verticillium 0 0 1 036 0 0 0 0Total 4 100 273 100 134 100 0 0

humans as well as animals and cause cutaneous fungalinfections [26 27] Parks are among the popular public placesfor the people to spend their time and have fun with theirfamily Our study revealed the presence of keratinophilicfungi such as dermatophytes in the soil of Shiraz parksM gypseum is a frequent geophilic dermatophyte commonlydistributed in soil worldwide Although nondermatophytefungi isolates were more common than dermatophytes in thepresent study M gypseum and M fulvum were the maindermatophytes which were isolated from four parks Theseparks potentially have a high risk for transmission of fungalinfections Previously these fungi were isolated from the soilsamples of different parts of Iran [28ndash31]

Some species of Aspergillus Fusarium solani and Bipo-laris spicifera are the causative agents of mycotic keratitis[32] Our study showed that the genus Fusarium was the firstdominant fungus in the soil of Shiraz which is in agreementwith the other studies conducted on the issue Moallaei et al[16 30] also reported that Fusarium was the most prevalentsaprophyte in South and Razavi Khorasan Provinces Thesecond most common species isolates in our study wereChrysosporium and Penicillium Chrysosporium species havebeen reported to be the causative agents of disseminateddiseases [33] Chrysosporium tropicum was reported fromcomb lesion in two different breeds of chicken in India [34]

In this study molecular method was utilized for iden-tification of keratinophilic fungi for the first time in Iran

Table 2 Distribution frequency of keratinophilic fungi isolatedfrom Shiraz parks soil

Species Number PercentFusarium spp 95 2308Fusarium chlamydosporum 4 097Fusarium oxysporum 3 082Fusarium solani 2 049Chrysosporium spp 54 1313Acremonium spp 52 1265Penicillium spp 51 1239Penicillium crustosum 2 049Penicillium palmae 1 024Aspergillus niger 40 973Aspergillus fumigatus 6 145Aspergillus sclerotiorum 2 049Aspergillus flavus 1 024Mucor spp 39 948Paecilomyces spp 17 413Microsporum gypseum 8 194Microsporum fulvum 2 049Bipolaris spicifera 5 121Bipolaris sp 1 024Bionectria ochroleuca 5 121Scedosporium apiospermum 3 082Scedosporium dehoogii 1 024Phialophora reptans 3 082Alternaria solani 2 049Alternaria alternata 1 024Cephalosporium curtipes 2 049Chaetomium spp 2 049Nectria mauritiicola 2 049Scopulariopsis sp 1 024Verticillium sp 1 024Malbranchea sp 1 024Tritirachium sp 1 024Ochroconis constricta 1 024Total 411 100

We could isolate and identify some genera of fungi such asBionectria spp which is important in natural products andmedicine The genus Bionectria is endophytic and has greatpotential for medicinal and agricultural applications [35]

Bionectria species are known as a destructive myco-parasite and grow inside the fungal host hyphae They areused as a biocontrol agent of plant-pathogenic fungi andare infrequently isolated from dead insects Besides theyare known as a parasite of living nematodes ticks andmyxomycetes [35 36]

Tritirachium species are an insect pathogenwhose naturalhabitats are soil and decaying plant materialsThese fungi areoccasionally isolated from head and nail infections [37 38]Furthermore Ochroconis spp are dematiaceous fungi thatcause deep mycoses such as chromomycosis around the


world There are many reports regarding the fungal diseasescaused by this fungus [39ndash41]

Scedosporium apiospermum is a common soil funguswith a worldwide distribution Environmental isolationshave been made from sewage sludge polluted streams andmanure of poultry and cattle In Australia Cooley et al [42]reported 59 cases of scedosporiosis caused by Scedosporiumapiospermum and S prolificans in patients with underlyingdiseases Invasive infections in normal patients are usuallycaused by traumatic implantation So fungal infections couldacquire or spread through playgrounds in parks

The current study investigated the relationship betweenthe frequency of fungi and the soils pH Bohme and Ziegler[19] reported the effect of the soil pH on the presence ofkeratinophilic fungi for the first timeMany researchers statedthat keratinophilic fungi could not be found in the soils withlow pH levels (3ndash45) Garg et al [3] also reported that theacidic soils with pH = 59 were free of keratinophilic fungiMoreover Asahi et al [43] demonstrated that keratinolyticenzymes were produced in pH of 6ndash9 and particularly theextracellular keratinase was active in pH = 9 In the presentstudy all the 411 keratinophilic fungi were isolated from thesoils with pH between 6 and 9 We found 6642 326 and097 keratinophilic fungi from the soil samples with pH of701ndash8 801ndash9 and 6ndash7 respectivelyThese findings have beenconfirmed by other studies as well Overall in this study 47of the places were contaminated with keratinophilic fungiand geophilic dermatophyte species isolated from the soilsof four parks These areas potentially have a high risk forcausing cutaneous fungal infections in humans and animalsand could be considered as a source of these infections

Acknowledgments

This study was supported by the Research Vice-ChancellorGrant no 6000 of Shiraz University of Medical SciencesShiraz Iran Hereby the authors would like to thank theResearch Improvement Center of Shiraz University of Medi-cal Sciences andMs A Keivanshekouh for improving the useof English in the paper

References

[1] S K Deshmukh and S A Verekar ldquoThe occurrence of der-matophytes and other keratinophilic fungi from the soils ofHimachal Pradesh (India)rdquo Czech Mycology vol 58 no 1-2 pp117ndash122 2006

[2] G Palsson ldquoGeophilic dermatophytes in the soil in SwedenStudies on their occurrence and pathogenic propertiesrdquo ActaVeterinaria Scandinavica supplement 25 pp 1ndash89 1968

[3] A P Garg S Gandotra K G Mukerji and G J F Pugh ldquoEcol-ogy of keratinophilic fungirdquo Proceedings Plant Sciences vol 94no 2-3 pp 149ndash163 1985

[4] M S Ali-Shtayeh and R M F Jamous ldquoKeratinophilic fungiand related dermatophytes in polluted soil and water habitatsrdquoin Biology of Dermatophytes and Other Keratinophilic Fungi RK S Kushawaha and J Guarro Eds pp 51ndash59 Revista Iberoa-mericana de Micologia Bilbao Spain 2000

[5] S K Abdullah and D A Hassan ldquoIsolation of dermatophytesand other keratinophilic fungi from surface sediments of theShatt Al-Arab river and its creeks at Basrah IraqrdquoMycoses vol38 no 3-4 pp 163ndash166 1995

[6] A K Gupta J E Ryder M Chow and E A Cooper ldquoDermato-phytosis the management of fungal infectionsrdquo Skinmed vol 4no 5 pp 305ndash310 2005

[7] S K Deshmukh Q A Mandeel and S A Verekar ldquoKeratino-philic fungi from selected soils of BahrainrdquoMycopathologia vol165 no 3 pp 143ndash147 2008

[8] S K Deshmukh and S A Verekar ldquoIncidence of keratinophilicfungi from the soils of Vedanthangal Water Bird Sanctuary(India)rdquoMycoses vol 54 no 6 pp 487ndash490 2011

[9] V Filipello Marchisio D Curetti C Cassinelli and C BordeseldquoKeratinolytic and keratinophilic fungi in the soils of PapuaNew GuineardquoMycopathologia vol 115 no 2 pp 113ndash119 1991

[10] R Papini F Mancianti G Grassotti and G Cardini ldquoSurvey ofkeratinophilic fungi isolated from city park soils of Pisa ItalyrdquoMycopathologia vol 143 no 1 pp 17ndash23 1998

[11] P Anbu A Hilda and S C B Gopinath ldquoKeratinophilic fungiof poultry farm and feather dumping soil in Tamil Nadu IndiardquoMycopathologia vol 158 no 3 pp 303ndash309 2004

[12] V M Ramesh and A Hilda ldquoIncidence of keratinophilic fungiin the soil of primary schools and public parks of Madras cityIndiardquoMycopathologia vol 143 no 3 pp 139ndash145 1998

[13] G M Vidyasagar N Hosmani and D Shivkumar ldquoKeratino-philic fungi isolated fromhospital dust and soils of public placesat Gulbarga Indiardquo Mycopathologia vol 159 no 1 pp 13ndash212005

[14] P A Volz M J Wlosinski and S P Wasser ldquoSparse diversity ofpotential pathogenic soil micro-fungi in the Ukrainerdquo Micro-bios vol 65 no 264-265 pp 187ndash193 1991

[15] M T Hedayati and M Mirzakhani ldquoSurvey of keratinophilicfungi in sewage sludge from wastewater treatment plants ofMazandaran Islamic Republic of Iranrdquo Eastern MediterraneanHealth Journal vol 15 no 2 pp 451ndash454 2009

[16] H Moallaei F Zaini M Pihet M Mahmoudi and J HashemildquoIsolation of keratinophilic fungi from soil samples of forestsand farm yardsrdquo Iranian Journal of Public Health vol 35 no 4pp 62ndash69 2006

[17] R Kachuei M Emami B Naeimi and K Diba ldquoIsolation ofkeratinophilic fungi from soil in Isfahan province Iranrdquo Journalde Mycologie Medicale vol 22 no 1 pp 8ndash13 2012

[18] K Pakshir and J Hashemi ldquoDermatophytosis in Karaj IranrdquoIndian Journal of Dermatology vol 51 no 4 pp 262ndash264 2006

[19] H Bohme and H Ziegler ldquoVerbreitung und keratinophilie vonanixiopsis stercoraria (hansen) hansenrdquo Archiv fur klinische undexperimentelle Dermatologie vol 223 no 4 pp 422ndash428 1965

[20] R Vanbreuseghem ldquoTechnique biologique pour Irsquoisolement desdermatophytes dusolrdquo Annales de la Societe Belge de MedecineTropicale vol 32 pp 173ndash178 1952

[21] S B Lee and J W Taylor ldquoIsolation of DNA from fungal my-celium and single cellsrdquo in Protocols A Guide to Methods andApplications M A Innis H D Gelfand J J Sninsky and TJ White Eds pp 282ndash287 Academic Press San Diego CalifUSA 1990

[22] T J White T Bruns S Lee and J Taylor ldquoAmplification anddirect sequencing of fungal ribosomal RNA genes for phyloge-neticsrdquo in PCR Protocols A Guide to Methods and ApplicationsA Innis D H Gelfand J J Sninsky and T J White Eds pp315ndash322 Academic Press San Diego Calif USA 1990


[23] S F AltschulW GishWMiller EWMyers and D J LipmanldquoBasic local alignment search toolrdquo Journal ofMolecular Biologyvol 215 no 3 pp 403ndash410 1990

[24] M V Fillipello ldquoKeratinophilic fungi their role in nature anddegradation of keratinic substratesrdquo in Biology of Dermato-phytes and Other Fungi R K S Kushawaha and J Guarri Edspp 77ndash85 Revista Iberoamericana de Micologia Bilbao Spain2000

[25] M D Connole ldquoReview of animal mycoses in AustraliardquoMyco-pathologia vol 111 no 3 pp 133ndash164 1990

[26] V FilipelloMarchisio L Preve andV Tullio ldquoFungi responsiblefor skin mycoses in Turin (Italy)rdquo Mycoses vol 39 no 3-4 pp141ndash150 1996

[27] R Spiewak and W Szostak ldquoZoophilic and geophilic dermato-phytoses among farmers and non-farmers in eastern PolandrdquoAnnals of Agricultural and Environmental Medicine vol 7 no 2pp 125ndash129 2000

[28] M A Zarei and M Zarrin ldquoIsolation of dermatophytes and re-lated keratinophilic fungi from the two public parks in AhvazrdquoJundishapur Journal of Microbiology vol 1 pp 20ndash23 2008

[29] G Dargahi The survey of pathogenic fungi and actinomycetesfrom soils of Ghazvin [PhD thesis] Laboratory Sciences Schoolof Medicine Tehran University of Medical Science TehranIran 1991

[30] T Shokohi M T Hedayati and H Bakhshi ldquoIsolation of fungiand aerobic actinomycetes from surface soil in Sarirdquo Journal ofKermanshah University of Medical Sciences vol 8 pp 25ndash322005

[31] S Shadzi M Chadeganipour and M Alimoradi ldquoIsolation ofkeratinophilic fungi from elementary schools and public parksin Isfahan IranrdquoMycoses vol 45 no 11-12 pp 496ndash499 2002

[32] M Eghtedari and K Pakshir ldquoAsyptomatic fungal cyst of con-junctiva caused byBipolaris spiciferardquo Iranian Journal ofMedicalSciences vol 31 no 1 pp 56ndash58 2006

[33] E Roilides L Sigler E BibashiH Katsifa N Flaris andC Pan-teliadis ldquoDisseminated infection due to Chrysosporium zona-tum in a patient with chronic granulomatous disease and re-view of non-aspergillus fungal infections in patients with thisdiseaserdquo Journal of Clinical Microbiology vol 37 no 1 pp 18ndash25 1999

[34] S A Saidi S Bhatt J L Richard A Sikdar and G R GhoshldquoChrysosporium tropicum as a probable cause of mycosis ofpoultry in Indiardquo Mycopathologia vol 125 no 3 pp 143ndash1471994

[35] W Ebrahim J Kjer M El Amrani et al ldquoTwo new peptidesfrom the endophytic fungus Bionectria ochroleuca isolated fromthemangrove plant Sonneratia caseolarisrdquoMarineDrugs vol 10pp 1081ndash1091 2012

[36] N C Paul J X Deng J H Lee and S H Yu ldquoNew recordsof endophytic paecilomyces inflatus and bionectria ochroleucafrom Chili Pepper Plants in Koreardquo Mycobiology vol 41 no 1pp 18ndash24 2013

[37] R N R Moraes M C T Ribeiro M C L Nogueira K C Cu-nha M M C N Soares and M T G Almeida ldquoFirst report ofTritirachium oryzae infection of human scalprdquoMycopathologiavol 169 no 4 pp 257ndash259 2010

[38] A Naseri A Fata and M J Najafzadeh ldquoFirst case of tritirac-hium oryzae as agent of onychomycosis and its susceptibility toantifungal drugsrdquoMycopathologia 2013

[39] D M Dixon and I F Salkin ldquoMorphologic and physiologicstudies of three dematiaceous pathogensrdquo Journal of ClinicalMicrobiology vol 24 no 1 pp 12ndash15 1986

[40] N Singh F Y Chang T Gayowski and I RMarino ldquoInfectionsdue to dematiaceous fungi in organ transplant recipients casereport and reviewrdquoClinical Infectious Diseases vol 24 no 3 pp369ndash374 1997

[41] K Singh J Flood R D Welsh J H Wyckoff III T A SniderandDA Sutton ldquoFatal systemic phaeohyphomycosis caused byOchroconis gallopavum in a dog (Canis familaris)rdquo VeterinaryPathology vol 43 no 6 pp 988ndash992 2006

[42] L Cooley D Spelman K Thursky and M Slavin ldquoInfectionwith Scedosporium apiospermum and S prolificans AustraliardquoEmerging Infectious Diseases vol 13 no 8 pp 1170ndash1177 2007

[43] M Asahi R Lindquist and K Fukuyama ldquoPurification andcharacterization of major extracellular proteinases from Tri-chophyton rubrumrdquoBiochemical Journal vol 232 no 1 pp 139ndash144 1985

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


from the superficial layer of the soil whose depth did notexceed 5ndash10 cm by using an iron spatula In doing so 300ndash400 gramof soil was collected in sterile polyethylene bags andbrought to laboratory for further processing

22 Measuring of Soil pH PH of each soil sample was meas-ured after preparation of soil suspension (one gram of soil tofive mL deionized water) using pH meter [17 19]

23 Isolation and Identification of the Isolates We used Van-breuseghemrsquos hair bait technique for isolation of keratino-philic fungi [20] Briefly each soil sample was thoroughlymixed and about 70 gram of the soil was packed in a sterile90mm Petri dish Then several pieces of sterile healthychildren hair fragments were dispersed over the surfaceof the soil samples and moistened with sterile distilledwater supplemented with antibiotic solutions chlorampheni-col (02 gmL) and strepto-penicillin (1000 IUmL) All thebaited soil Petri dishes were incubated at room temperature(20ndash25∘C) in the dark for 2 months and got moistened ifnecessary After observation of colony growth around thehairs the colonies were subcultured on Sabouraudrsquos dextroseagar (SDA)with andwithout chloramphenicol (50mgL) andcycloheximide (500mgL) and purifiedThe fungi were iden-tified based on the conventionalmethod (colonymorphologyand macro- and microconidia characteristics) and DNA-based identification techniques

24 DNA-Based Identification Techniques (DNA SequenceAnalysis) Molecular identification of the unknown isolateswas achieved by DNA sequence analysis First the fungi weregrown in flasks containing Sabouraudrsquos dextrose broth andincubated at 25∘C for several days using shaker rotator Aftercolony growth the culture was filtered (Millipore USA) andthe fungal mass was washed with distilled water for severaltimes and stored in a freezer (minus20∘C) for further processing

DNAwas extracted by using Lee technique [21] withmildmodification First the frozen mycelium mass was smashedby mechanical pressure using sterile pounder and liquidnitrogen The acquired powder was then mixed with lysesbuffer and the DNA was extracted The ITS1ndash58SndashITS 2rDNA was amplified using ITS1 and ITS4 as forward andreverse primers as described by White et al [22] Amplifi-cation was performed in 50 120583L reaction volumes containing5 120583L of 10times buffer MgCl

2(25mm) 15 120583L dNTP (10mM)

05 120583L 05 120583L of each 02Mm primer (ITS1 31015840-TCC-GTA-GGT-GAA-CCT-GCG-G-51015840 and ITS4 31015840-TCC-TCC-GCT-TAT-TGA-TAT-GC-51015840) TaqPolymerase (125U) 05 120583LDNAsample 1 120583L and distilled water 405 120583L The PCR reactionwas carried out using a Thermal Cycler (R corbet cg1-96)with the following conditions denaturation at 94∘C for 5min34 cycles of (30 s at 94∘C 45 s at 56∘C and 45 s at 72∘C)extension at 72∘C for 7min and storage at 4∘C Negativecontrols were also used in each set of reactions The finalproducts were analyzed by electrophoresis on 12 agarosegel (Sigma) and stained with 05120583gmLminus1 ethidium bromideIn addition the PCR products were sent for sequencingin both directions (Bioneer Korea) The sequence results

Figure 1 Agarose gel electrophoresis and PCRproducts bands lines1 and 2 (marker) lines 3ndash12 (up) and 2ndash8 (down) are PCR productsof many unknown fungi

were processed by using the web-based blasting programbasic local alignment search tool (BLAST) at the NCBI site(httpwwwncbinlmnihgovBLAST) and the data werecompared with the NCBIGenebank database [23]

25 Data Analysis The study data were analyzed throughFisher exact test and Chi-square test

3 Results

PCR products bands on gel agarose are presented in Figure 1From the 196 soil samples a total of 411 colonies of ker-atinophilic fungi were isolated from 43 parks The fun-gal isolates belonged to 22 genera as follows Fusarium(2530) Penicillium (1313)Chrysosporium (1313)Acre-monium (1265) Aspergillus (1192) Mucor (948) Pae-cilomyces (413) Microsporum (242) Bipolaris (145)Bionectria (121) Pseudallescheria (073) Phialophora(073) Alternaria (073) Nectria (048) Cephalospo-rium (048) Chaetomium (048) Scopulariopsis (024)Scedosporium (024) Verticillium (024) Malbranchea(024)Tritirachium (024) andOchroconis (024)Moredetails about the species are presented in Table 1

Fusarium spp was the most common fungal isolateamong the parks Besides eight species of Microsporumgypseum were isolated from four parks with soil pH betweenseven and nine and two species ofMicrosporum fulvum fromone park with the same pH Most of the fungi were isolatedfrom the soil samples with pH between 7 and 8 (6642) andno colony growthwas seen in pH gt 9 as shown in Table 2Thestudy results revealed no significant correlation between soilpH and fungal speciesMore details about the isolates and soilpH are presented in Table 2

4 Discussion

The keratinolytic activity of keratinophilic fungi is importantfor ecology and has attracted many researchersrsquo attentionaround the world Keratinophilic fungi play an importantrole in the natural degradation of keratinized residues in thesoil [24 25] Some types of these fungi such as geophilicdermatophytes live in soil and could be transmitted to



Fungal genuspH

6-7 7-8 8-9 gt9119899 119899 119899 119899














Acknowledgments


References




















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



Fungal genuspH

6-7 7-8 8-9 gt9119899 119899 119899 119899














Acknowledgments


References




















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology





Acknowledgments


References




















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology






























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

Research Article Isolation and Molecular Identification of … · 2019. 7. 31. ·...

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