Incidence of keratinophilic fungi in areas of Raipur City , Chhattisgarh region , India

In an endeavor to search for some wild isolates of keratinophilic fungi from the Raipur City of Chhattisgarh, 24 soil samples from different regions of the city were collected and screened using hair baiting technique. A total of six species which belong to two genera were isolated. They are Chrysosporium keratinophilum (27.78%), Chrysosporium pannicola (5.56%), Chrysosporium tropicum (13.89%), Chrysosporium sp. (close to Chrysosporium pannicola) (11.11%), Chrysosporium sp. (16.67%) and Microsporum gypseum (25%). The Chrysosporium species prevalent in this region are able to tolerate temperature of about 40°C and all isolates belong to moderate individual risk group.


INTRODUCTION
Soil is the main reservoir of different types of fungi and some of them are pathogenic to both humans and animals.Soil is also a good source of keratinophilic fungi and the probability of the incidence of such fungi increases manifold if it is rich in keratinous materials (Marchisio, 2000).Keratinophilic fungi, also known as dermatophytes, are mostly pathogens of humans and other animals but also have the ability to live saprotrophically.Every keratinophilic fungus has the potential to cause infection and tissue invasions (Simpanya, 2000).
In recent past, many investigations have been carried out to find the distribution pattern of the keratinophilic fungi in many parts of India (Jain and Sharma, 2011;Singh et al., 2009;Sharma et al., 2008;Anbu et al., 2004;Deshmukh, 2002Deshmukh, , 2004;;Ghosh and Bhatt, 2000;Kaul and Sumbali, 1997).Barring a few reports, this area of Chhattisgarh remained unexplored for the occurrence of keratinophilic fungi (Khanam et al., 2002).Hence, it was felt significant to screen soil of different areas of Raipur City, in order to evaluate the extent and presence of keratinophilic fungi in the environments.The study was undertaken at two potential sites viz.poultry farm and hair-dumping/garbage area and the results obtained are reported.

MATERIALS AND METHODS
Soil samples were collected from the superficial layer at a depth of 3-6 cm of four poultry farms and six hair-dumping/garbage sites from different areas in and around Raipur city.The soil samples were placed in sterile polythene bags, brought to the laboratory and stored overnight at 4°C.Approximately 18 to 20 g of soil from each sample were placed in 90 mm sterile Petri plates in five replicates.Short (1-2 cm length) sterilized defatted human hair fragments were scattered on the surface of soil for baiting.The plates were moistened with an antibiotic solution containing cycloheximide (0.5 mg/ml) and chloramphenicol (0.05 mg/ml).The plates were incubated at room temperature (26±2°C) for a period of 4 to 6 weeks and remoistened with sterile deionized water periodically.The plates were examined daily under a stereoscopic binocular microscope and if growth was observed then the baits were selected at random from each Petri plate and transferred to plates containing Sabouraud Dextrose Agar (SDA) medium supplemented with cycloheximide (0.5 mg/ml) and chloramphenicol (0.05 mg/ml).The SDA plates were incubated at room temperature (26±2°C) for *Corresponding author.E-mail: toashis@gmail.com.further examination.Fresh developed colonies were examined and identified following the key proposed by Chabasse (1988), von Oorschot (1980) and Carmichael (1962).The identification of isolates was further confirmed by NFCCI (Pune) and IMTECH (Chandigarh).

RESULTS
A total of 24 soil samples were examined from poultry farm and hair-dumping/garbage sites.Out of them nine samples (37.5%) were positive for keratinophilic fungi (Table 1).

Distribution of keratinophilic fungi in poultry farm soil
Distribution of keratinophilic fungi in poultry farm soils around Raipur is presented in Figure 1.A total of 14 colonies of keratinophilic fungi were isolated from five soils samples.Four species belonging to two genera were observed in poultry farm soil.Only one dermato-   keratinophilum (22.73%), C. tropicum (13.64%) and C. pannicola (9.09%) (Figure 2).Other than keratinophilic fungi different species of the fungal genera viz.Aspergillus, Penicillium and Fusarium were observed.

DISCUSSION
Keratinophilic fungi are very closely related to dermatophytes, having the ability to cause infection.They parasitize hard keratin tissues and can also invade and degrade them.Thus, they are keratinophilic and keratinolytic in nature.Soils enriched with keratin substrate like hairs, feathers, horn and hoofs, skin etc. are good reservoir of dermatophytes as well as keratinophilic fungi (Marchisio, 2000;Ajello, 1974).Dermatophytes are classified into three genera namely Epidermophyton, Microsporum and Trichophyton (Summerbell, 2000).Other than this, closely related species includes 100 genera like Chrysosporium, Malbranchea, Geomyces, Scopulariopsis, Doratomyces and many others having keratinolytic ability (Blyskal, 2009).Kushwaha (2000) reviewed the presence of 47 species, a few of them with uncertain positions, among the genus Chrysosporium throughout the globe.
In the present study, the only dermatophyte isolated was M. gypseum (25%), the most common species reported amongst the dermatophytes.It is one of the most prevalent soil borne species and a major causative agent of infection to human and animals (Simpanya, 2000).Among the closely related species the occurrence of C. keratinophilum (27.78%), C. tropicum (13.89%) and C. pannicola (5.56%) were noteworthy in this region.These species are thermotolerant and can grow considerably at 35 to 40°C; the exceptional one is C. tropicum which can withstand temperature upto 45°C (Ellis et al., 2007;van Oorschot, 1980).The high occurrence of keratinophilic fungi especially Chrysosporium sp. in the soil of Raipur region support the fact that these species are able to withstand the high temperature of this region which is about 45 ± 2°C in summer.
The prevalence of fungal infections in the region as reported routinely in district hospital and Medical College of Raipur showed that 66.7% infective patients belong to urban areas and 33.3% from rural areas.Superficial fungal infection by various species of Trichophyton was reported to be highest (89%) followed by Microsporum sp.(11%).The reported disease types most prevalent in this area are tinea corporis (44.5%) followed by tinea pedis (22.3%).
Among other types of tineas, tinea capitis, tinea cruris and tinea manuum have also been reported occasionally in this region (Pradhan, 2012).In any region, tinea corporis and tinea pedis are the most common types of infection and all types of dermatophytes have the potential to cause tinea corporis.Tinea pedis is possibly caused by Trichophyton rubrum, Trichophyton mentagrophytes var.interdigitale and Epidermophyton floccosum (Degreef, 2008).Apart from these anthropophilic dermatophytes mentioned above the present study revealed that this part of India has good presence of geophilic and keratinophilic Chrysosporium and Microsporium species, which could add up to the increase in dermatophytic infection in this region.

Figure 2 .
Figure 2. Distribution of fungi in hair dumping/garbage soil.

Figure 4 .
Figure 4. Percent distribution of keratinophilic fungi in areas of Raipur City.
infective microorganisms are classified into four risk groups viz.RG -1 to 4. It is based on pathogenicity of the organism, modes of transmission and host range of the organism.RG-1 having no or low individual and community risk whereas RG-2 have the moderate individual risk and low community risk.RG-3 shows high individual risk but low community risk and RG-4 have high individual and community risk.Following the classification criteria of WHO, Australia, Canada, European Union (EU), USA and other nations has placed Microsporum sp. in RG-2 (American Biological Safety Association, 2013).Other than M. gypseum, all isolated Chrysosporium sp. were geophilic and keratinolytic, often recovered from skin and nail scrapings of feet.Some of the Chrysosporium sp.closely resembles true deramatophytes like Trichophyton and Microsporum (van Oorschot, 1980).Based on pathogenicity, Chrysosporium sp. has been placed in RG-2 (Table

Table 1 .
Distribution of soil sample examined.

Table 2 .
Species wise characteristics and pathogenicity.