07_chapter2.pdf

REVIEW OF LITERATURE It was Sabouraud (1893), who expressed an opinion that soil may represent the main reservoir of fungi and postulated that dermatophytes survive in the soil as saprophytes. But the early failure to recognize the existence of soil keratinophilic fungi was due to lack of selective isolation techniques for these fungi. The introduction of Vanbreuseghem's (1952) hair-bait

method,

more

precisely the To-Ka-Va

hair baiting

method

(Benedek, 1962) for the isolation of keratinophilic fungi from the soil marked an important step ahead in broadening our knowledge of keratinophilic fungi.

It led not only to the recognition of soil as a basic substrate for these fungi, but also the long predicted natural saprophytic stage of dermatophytes in the soil · was investigated and reviewed throughout the world by several workers

(Ajello,

1960, 1974 and

Piontelli and Caretta,

1974). Their

discoveries opened an era of study of geophilic keratinophilic fungi present in the soil.

DISTRIBUTION OF KERATINOPHILIC FUNGI The significance of ecology of keratinophilic fungi with special respect to dermatophytes has become a subject of enquiry by a large number of workers ever since their geophilic nature was established (Pugh and Mathison, 1962; Ajello and Alpert, 1972). Keratinophilic fungi are a small but well-defined and important group of fungi that degrade hard keratin. Throughout the world great interest is shown by research workers in soil mycoflora that can degrade keratinized residues. This is due to two factors, the extreme resistance of keratin to biological attacks and the pathogenic potential of keratinolytic saprophytic species. According to their natural habitats, keratinophilic fungi are divided into 3 categories anthropophilic, zoophilic and geophilic, therefore include both several of the most important dermatophytes and saprophytic species. Most of the keratinophilic fungi are not dermatophytes but are soil inhabitants. Keratinophilic saprophytes are found in soil enriched with keratin. They occur on cornified debris in the soil and degrade keratin and keratinous material. Therefore, they play an important ecological role decomposing such residues. Keratinolytic fungi may be important ecologically especially where human and animal population exert strong selective pressure on the environment. In fact, the distribution of such fungi seems to be ecologically restricted to keratin material available 17

as a substrate in the environment. Several surveys have shown that the distribution in soil of dermatophytes and keratinophilic fungi is influenced by the enrichment with keratin due to high concentration of people and of wild and domestic animals (Baggy, 1986; AIi-Shtayeh Musallam, 1990; Papini

et al., 1988a, b; AI-

et al., 1998 and Ramesh and Hilda, 1999). Griffin

(1960) pOinted out that keratinophilic fungi are restricted to keratinous substrates present in the soil and that keratin is the major limiting factor contributing to their distribution. Subsequent surveys led to the conclusion that enrichment of soil with keratinous material provides a most conducive habitat for the growth and occurrence of keratinophilic fungi (Padhye 1966 and Mercantini

et al.,

et al., 1980, 1983). Hence, it proved that the soil rich in

keratinic residues constitute a permanent or occasional

reservoir for

dermatophytes, keratinolytic and keratinophilic fungi and is a source of potential infection for man. The potential pathogenecity of keratinophilic fungi (Rippon, 1982) has been considered as a natural evolution from keratin utilization in the soil (geophilic species) to invasion of cornified substrates in animals (zoophilic species) and men (anthropophilic species) (Marchisio and Luppi Mosca, 1982). Research show that the distribution in soil of dermatophytes and other keratinophilic fungi is influenced by the organic content and in particular by the enrichment of soil with various keratin sources. The majority of dermatophytes, even the most specialized do not depend totally on the presence of host for survival as they can grow saprophytically. Soil has therefore, been thought of as a link in the complex epidemiological chain that relates in both the evolutionary and developmental sense these ecological groups geophilic, zoophilic and anthropophilic dermatophytes together (Marchisio, 1986). In general the habitats mapped out and reported to be rich in these fungi are the soils from densely populated areas, inhabiting a variety of ecological sites frequented by animals and men such as public parks, play grounds, poultry farms, crop fields, cattle yards, stables, zoological parks, swimming

pools,

recreation

centers

and

other places frequented

mammals,

birds and decomposed organic matter (AIi-Shtayeh,

by

1989;

et al., 1989; AI-Musallam, 1990; Soon, 1991; Kaul and Sumbali, 1994; Ulfig and Korcz, 1995 and Ulfig et al., 1996). In whatever habitat they

Mercantini

occur, they are a group of botanically related fungi with affinity for epidermal 18

scales and hairs. Mammals and birds thus create suitable habitat for the saprophytic growth of keratinophilic fungi in soil. Their hair, wool, horn, hoof, feather constitute natural baits for the isolation of such fungi either from animal bodies or from various habitats where they are found (Abdel Fattah

et al., 1982; Calvo et al., 1984; Baggy and Abdel Hafez, 1985; Baggy, 1986; Ali-Shtayeh et al., 1988a, band AI-Musallam, 1990). Hence, public parks, open playgrounds, crop fields that are often invaded by mammals and birds provides suitable habitat for the survival of keratinophilic fungi. These visitors leave their reSidues, which probably contaminate the soil with fungal propagules.

Kaul

and Sumbali (1999a) studied the ecological factors

governing the distribution and survival of keratinophilic fungi in poultry farm soils. All poultry farm soils were rich in humus and the keratinophilic fungi were generally found to be proportional to the. soil organic matter. These soils were nearly neutral to weakly alkaline and organically rich with a high content of organic carbon, nitrogen, phosphorus, potaSSium, magnesium, calcium and iron. Information on the existence of such fungi is available from the dry habitat of desert soil (Baggy, 1992) to the adverse cold climatic conditions of Antarctic (Mercantini

et al., 1989). Isolation of keratinophilic

fungi from Galapagos Island conducted by Ajello and Padhye (1974) was interesting because the collection sites were unusual, the xeric low lying coastal areas of the islands and the soil was essentially made up of diSintegrated lava, low in organic matter. Randhawa and Sandhu (1965) pOinted out that saline soils are poor habitats for keratinophilic fungi. However Padhye

et al. (1967) reported their occurrence in marine soil of

Bombay but these fungi could not be encountered in the salt marshes of the coastal Mediterranean areas in the recent study of Abdel Fattah

et al. (1982)

whereas Katiyar and Kushwaha (1997) isolated keratinophilic fungi from Mediterranean Sea beach. Caretta and Piontelli (1975) commented that the isolation of these fungi from habitats with non-keratinous substrates might represent only a mean of their dispersal. Besides soil, birds nest (Sur and Ghosh, 1980b), hairs of wild animals (Baggy and Abdel Hafez, 1985; Baggy, 1986; AIi-Shtayeh

et al., 1988a, b,

1989 and Simpanya and Baxter, 1996a), skin and pens of animals (Efuntoye and Fashanu, 2001a), house dust (Katiyar and Kushwaha, 2002), water (Mangiarotti and Carretta, 1984), plant debris and dung (Currah, 1985), sewage sludge (Ulfig

et al., 1996), coal mines (Ulfig and Korcz, 1995), 19

school floors (Mercantini et al., 1986; AIi-Shtayeh and Asa'd AI-Sheikh, 1988 and Ali-Shtayeh and Arda, 1989), children sand pits (Marchisio and Luppi Mosca, 1982; Marchisio, 1986 and Ali-Shtayeh, 1988) are other different ecological

nitches

for

the

survival

of

potential

dermatophytes

and

keratinophilic fungi. All these studies provide evidence that the distribution of such fungi depends on the amount of keratin material available due to the presence of mammals and birds. Their droppings hair, wool, nail, feather, horn, hoof, skin scraping, scales and other cornified epidermal tissues serve as natural substrate for the growth and survival of these fungi. Various forms of keratin have been used in cultures to estimate their potential to serve as substrate for keratinophilic fungi. Kushwaha and Agarwal (1976) isolated keratinophilic fungi from various substrates like horn, hair, nail, feather and wool. There are several reports showing that a variety of keratinophilic fungi are present on the hairs of wild animals without causing any apparent lesions (Okafar and Gugnani, 1981; Baggy and Abdel Hafez, 1985; Baggy, 1986 and AliShtayeh et al., 1988a, b, 1989). Knudsen (1980) has reported the survival of

Epidermophyton

flocossum,

Microsporum

canis,

Trichophyton

mentagrophytes and T. rubrum on skin scrapings. Mercantini et al. (1980) while surveying soils of public gardens and parks in Rome found that keratinous substrates in soil definitely influences the biological cycle of these fungi. Fungi have been isolated from the skin of different animals in different part of the world (Garcia-de-Loma et al., 1980; Garcia et al., 1981 and Marsella et al.,

1985). AIi-Shtayeh et al.

(1988a,

b,

1989) isolated

keratinophilic fungi on hairs of goats, cows, cats, dogs, donkey and sheep respectively from west bank of Jordan. Rai and Qureshi (1994) screened different keratin baits to isolate keratinophilic fungi. Simpanya and Baxter (1996a) isolated fungi from the pelage of cats and dogs. Mitra et al. (1998) carried out a survey in different areas of Uttar Pradesh for isolating dermatophytes from ruminants and studied 102 samples of skin scrapings collected from various animals. Efuntoye and Fashanu (2001a) determined the mycoflora of skin scrapings, hairs, nails and pens of healthy animals in Nigeria. Maghazy (2001) isolated dermatophytes and keratinophilic fungi on healthy children's hairs and nails. Animals, be large or small serve as an important source of secondary infection of dermatophytoses in men. It may be concluded from these 20

observations that wild animals not only act as a carrier of dermatophytic fungi but also provides a suitable habitat for their survival as saprophytes. It is evident from the above account that keratinophilic fungi and allied derrmatophytes survive saprophytically in soil and on wild animals, including birds and humans, which provide an inoculum for primary and secondary infection to various animals and humans. Various ecological factors affect and control their survival, distribution, pathogenesis and

keratinolytic

activity. These animals may thus act as a direct source of infection, when in contact with them or as an indirect source of infection by contaminating the working areas and dwelling places (Rippon, 1982). Hence, animals play an important role in the transmission and persistence of dermatophytes and other potentially pathogenic fungi (Zaror et al., 1986 and Caprilli et al., 1987) and act as a vector for these diseases (Rippon, 1982; Martinez-Roig and Torres Rodriguez, 1986 and Ogbonna et al., 1986). Thus, it becomes evident that animalization, that enrichment of soil with keratinous organic matter creates suitable conditions for the growth of keratinophilic fungi. Similarly birds, their pelage and feathers are also related with the occurrence, distribution and transport of pathogenic dermatophytes and keratinophilic fungi. They act as mechanical career of some disease agents. Emmons (1955) established the first link between birds and pathogenic fungi when he isolated Cryptococcus neoformans from pigeon droppings. Since then keratinophilic fungi and some related dermatophytes have been occasionally recorded on bird's feather. Pugh (1966) conducted the first survey of keratinophilic fungi on Indian birds in Madras. Sur and Ghosh (1980b) isolated. keratinophilic fungi from Passeriformes and Galliformes from Orissa. Other global surveys have also shown that the Passeriformes birds particularly the species Passer domesticu5, carries maximum number of fungi (Sur and Ghosh, 1980b and Humpolichova and Otcenasek, 1981). Sarangi and Ghosh (1991) reported keratinophilic fungi inhabiting Passer domesticus in two districts of Orissa. Dixit and Kushwaha (1991) isolated 13 species of keratinophilic fungi from 87 free-living Indian birds. Camin et al. (1998) isolated keratinophilic fungi from feathers of 110 starlings (Sturnus vulgaris)

in

Brittany, France.

Kaul and Sumbali

(2000) also isolated

keratinophilic fungi from feathers of Indian poultry birds. Efuntoye and Fashanu (2001b) recovered 15 species of fungi from feathers, nails and beaks of 120 common birds in Nigeria. The occurrence of these fungi on 21

free-living

birds indicates that they

may play an

important role

in

dissemination of fungi. In addition to the availability of keratinous substrates in soil, the frequency of occurrence and survival of keratinophilic fungi in the soil is also influenced by a number of ecological factors both abiotic and biotic (Otsenasek, 1978 and Garg

et al., 1985). Among abiotic factors, physical

factors of importance are soil pH, soil moisture, chemical composition and quantity of organic matter in the soil. The effect of soil pH on these fungi has been first observed by Bohme and Ziegler (1965), the physiology of decomposition of keratin over a wide range of pH was also given by Ziegler (1966). Several workers (Ajello and Padhye, 1974; Abdel Fattah

et al.,

1982; Schon born, 1983 and AI-Musallam, 1989) reported the occurrence of keratinophilic fungi in soil with high (5.0%) or low (1.5%) humus content. The distribution of certain keratinophilic species was found to be selective in relation to soil humus, while others were distributed irrespective of soil humus (Chmel

et al., 1972; Chmel and Vlacilikova, 1977 and Schonborn,

1983). The occurrence and survival of keratinophilic fungi has reported to be influenced by various genomic and climatic factors such as chemical composition of organic matter in soil, its quantity as a source of nutrition and energy, soil humidity, pH, temperature, depth of soil profiles, soil texture and structure. Chmel

et al. (1972) found their presence to be directly

proportional to the humus content of soil. Chmel and Vlacilikova (1975) studied the influence of depth and humus content of soil on the occurrence of keratinophilic fungi and found their highest frequency in the superficial layer of soil. Nigam and Kushwaha (1990b) also studied the existence of keratinophilic fungi with emphasis on Chrysosporium and indicated that reports of less number of Chrysosporium species from India may be due to less attempt of their isolation from

different horizons.

Existence of

keratinophilic fungi in the soil is also influenced by the presence of other constituents

of

bio-cenotic

complexes,

mainly

the

bacteria

and

actinomycetes that exert antagonistic effect on keratinophilic fungi (Nigam, 1987 and Dixit, 1991). Other factors that influence the occurrence of these fungi are moisture content and aeration of surface soil layers. In studies of Randhawa and Sandhu (1965); Garg (1966c) and Govil

et al. (2001) a

comparison in the prevalence of keratinophilic fungi in the hilly areas of Jammu and plains of Rajasthan and hilly areas of Shimla and plains of Agra 22

has been demonstrated which clearly indicates that maximum distribution of keratinophilic fungi appeared in hilly areas when compared to plains. The difference in the prevalence of keratinophilic fungi in the soil of different parts of the India may be due to some distinction in the climatic conditions. Garg (1966c) also emphasized that the climate and perhaps other environmental factors are apparently important in determining the distribution of keratinophilic fungi. Chmel

et al. (1972) also observed that

atmosphere, humidity, temperature, nature and reaction of soil play an important role in their passage from saprophytism to parasitism. Currah (1985) stated that potentially pathogenic keratinophilic fungi have developed various structural modifications in response to a reliance on animals for habitat formation and propagule dissemination. For the keratinophilic fungi, which are zoophilic dermatophytes, extra human environment represent only the substrate in which they may survive for a certain time but not live an active life. The capability of long-term existence on host depends on complex relation between them.

Worldwide distribution After dealing with different aspects of distribution of keratinophilic fungi, the conclusion inferred from the ecological studies carried out in different areas of the world, that the prevalence of these fungi varies in different regions 'of the country exhibiting geographical specialization. Ajello (1974) studied the occurrence of many fungi in soil of different countries and gave an account of natural history of dermatophytes and their geographical distribution (Ajello 1960; 1974 and Vanbreuseghem 1970). In subsequent years researchers from several countries reported their occurrence in soil as enumerated below, proving their geophilic nature: Antarctic (Mercantini

et

al., 1989, 1993), Arctic soil (Currah et al., 1996), Australia (Mc Aleer, 1980), Brazil (Chatanon et al., 1994), Eastern Islands (Ajello and Alpert, 1972), Egypt (Abdel Fattah et al., 1982; Abdel Hafez, 1991; Baggy 1992; Moubahsen et al., 1992 and Youssef et al., 1992), France (Chabasse, 1988; Chabasse et al., 1989 and Agut et al., 1995), Galapagos Island (Ajello and Padhye, ' 1974), Gottingen (Meissner and Quadripur, 1983), Iraq (Abdullah

et al., 1986), Italy (Marchisio and LuppiMosca, 1982; Marchisio, 1986; Marsella and Mercantini, 1986; Marchisio et al., 1991; Carreta et al., 1992; Manchianti and Papini, 1996 and Papini et al., and Hassan, 1995), Israel (Alteras

1998), Jordan (Ali-Shtayeh, 1988, 1989; Ali-Shtayeh and Arda, 1985, 1989; 23

Ali Shtayeh and Asa'd AI-Sheikh, 1988), Kenya (Muhammed and Lalji, 1978), Kuwait (AI-Musallam 1989, 1990 and AI-Musallam

et al., 1995), Malaysia

(Soon, 1991), New Zealand (Allred, 1982; Simpanya and Baxter, 1996b), Nigeria (Ogbonna and Pugh, 1987), Portugal (Cabrita

et al., 1984), Rome

(Mercantini et al., 1980, 1983, 1986; Marsella et al., 1985 and Caprilli et al., 1987), Russia (Volz et al., 1991, 1993), Spain (Guarro et al., 1981; Calvo et

al.,

1984;

Torres-Rodriguez et al.,

1986 and

Cano

et al.,

1987),

Mediterranean Sea beach, Spain (Katiyar and Kushwaha, 1997a), Signey Island, South Orkney Island (Pugh and Allsopp, 1982) and Yemen (EI-Said, 1994; EI-Said and Abdel Hafez, 1995).

Indian Reports In India, the first report of the occurrence of Microsporum gypseum in the soil was from the vicinity of Dibrugarh district of Assam (Dey and Kakoti, 1955). Later, Randhawa and Sandhu (1965), Garg (1966c) and Gugnani and Randhawa

(1973) performed the survey of these fungi

and gave a

comprehensive account of distribution of keratinophilic fungi in Indian soil. The above workers have laid more emphasis on the soil of Jammu and Kashmir, Delhi, Rajasthan and U.P. Padhye

et al. (1966, 1967) emphasized

on the soil of Poona and Bombay. Deshmukh and Agarwal (1983a) isolated some keratinophilic fungi from coastal habitats of Goa.

Deshmukh (1999)

and Deshmukh et al. (2000) isolated keratinophilic fungi from the soil of Mumbai and Mysore respectively. Sunderam (1987) reported keratinophilic fungi from rice fields of Madras. Latha

et al. (1992) isolated them from Tamil

Nadu . Ramesh and Hilda (1999) surveyed keratinophilic fungi in the soil of primary schools and public parks of Madras city. Dixit and Kushwaha (1990a) studied soil of Andaman Islands. Roy et al. (1972) and Sur and Ghosh (1980a) have worked on the soil of Orissa. Sarangi and Ghosh (1991) isolated keratinophilic fungi from Orissa. Ghosh and Bhatt (2000) isolated keratinophilic fungi from Chilka lake site, Orissa.

Jain and Agarwal (1977,

1979) isolated keratinofers from the soil of Mt. Abu. Singh

et al. (1994,

1996) isolated some keratinophilic fungi from Bharatpur Bird's sanctuary (Rajasthan). Verma et al. (1982) have isolated some keratinophilic fungi from soils of Bihar. Kaul and Sumbali (1994) emphasized on the prevalence of keratinophilic fungi in dairy farm soil of Jammu. The same workers (1999a, 2000) isolated keratinophilic fungi inhabiting poultry farm soil of Jammu. 24

Kushwaha and Agarwal (1976); Deshmukh and Agarwal (1983b); Jain (1983); Singh and Agarwal (1983); Agnihotri and Agarwal (1989); Hasija et

al. (1990) and Jain et al. (1993) worked on the soil of M.P. Singh and Agarwal (1983) studied this group of fungi intensively in Central India particularly in Sagar, Patharia, Chattarpur, Bizawai, Tikamgarh and Gwalior region. Rajak et al. (1989) isolated soil keratinophilic fungi from gelatin factory campus in Jabalpur. Sharma et al. (1997) isolated them from Betul. Khanam and Jain (2002) isolated keratinophilic fungi from the soil of Damoh. Keratinophilic dermatophytes were also isolated from the hilly areas of U.P (Padhye and Thirumalachar, 1962 and Deshmukh, 1985) and plains around Kanpur (Nigam, 1987 and Dixit, 1991). The keratinophyles were also isolated from the Indo-Gangetic plains and densely populated areas of U.P. (Jain et al., 1985). Jain et al. (1985) isolated keratinophilic fungi from Lucknow. Nigam and Kushwaha (1989d, 1990b) carried out a detailed study of the occurrence of keratinophilic fungi and non-keratinophilic fungi in house dust of Kanpur. Gupta and Garg (1991) isolated keratinophilic fungi from Meerut. Awasthi and Gotewal (1991) studied keratinophilic flora of Bareilly. Singh et al. (1990) also isolated keratinophilic fungi from Agra soil. Singh et al. (1995) isolated some strains of Microsporum gypseum from Agra soil. Govil et al. (2001) compared the diversity of keratinophyles in the soil of Simla

and

Agra.

Saxena

et al.

(2004)

studied

the

diversity of

keratinophilic fungi in the soil of Agra. All these reports from India and abroad justified the worldwide distribution of keratinophilic fungi proving their cosmopolitan nature.

TAXONOMY Since keratinophilic: fungi were found associated with the diseases of the cutinized and keratinized tissues of men and animals, they were found to possess proteolytic enzymes, capable of degrading keratinized substrates, it possess morphological similarities and existence with dermatophytes in natural conditions, developed interest and initiated people to look into this group. The enhancement of the spectrum of dermatophytes by primary soil fungi, which may act as agents of dermatophytoses relatively rarely and under special circumstances only brought a · need of classification of dermatophytes according to the basic manner of heterotrophy. The geophilic dermatophytes

and

keratinophilic

fungi

are

thought

to

be

primitive

representatives as the ecological criteria have become the basis of evolution 25

theory according to which dermatophytes have gone through a long term process of biological differentiation and specialization (Otcenasek, 1978). Emmons

(1934)

laid

down

the

foundation

of

taxonomy

of

dermatophytes. The identification of Trichophyton and Microsporum species on the basis of their micro-conidia was problematic. Most species occurred as dermatophytes on men and animals. Many names were applied on the basis of clinical aspects only; description of the micro-morphology of the fungi was often omitted or vague. Copious superfluous names were allotted to species of the two genera and the overall picture became rather confused. Ajello (1968), Caprilli et al. (1971) and Emmons et al. (1977), revised the genus Trichophyton and Microsporum reducing a large number of species to synonyms. Corda (in Sturm, 1833) introduced the name Chrysosporium for a single species C.

corii.

Saccordo (1901)

placed

it in synonym with

Sporotrichum. Later Carmichael (1962); Dominik (1967); Van Oorschot (1980)

and

Kushwaha

(1985,

1987,

1994)

reviewed

this

genus.

Chrysosporium was considered related to the genera Trichophyton and Microsporum, which have Chrysosporium like micro-conidia in addition to macro-conidia. A characteristic feature that macro-conidia production often declines

with

repeated

sub-culturing;

leaving

only

micro-conidia

is

noteworthy in these genera. The genus Chrysosporium was isolated in most of the soil surveys in India and abroad showing high percentage of occurrence (Carmichael, 1962; Randhawa and Sandhu, 1965; Garg, 1966a, b; Van Oorschot, 1980; Jain et al., 1985 and Nigam and Kushwaha, 1990b). The species of Chrysosporium are described mainly on the basis of the size of their conidia (Carmichael, 1962 and Van Oorschot, 1980). The taxonomy of Chrysosporium is based on the size of the conidia, broad basal scar of conidium and colony morphology. Certain other characters were also considerable from time to time for their differentiation. Padhye (1969) advocated an additional character of thermo-tolerance in this genus for the differentiation oF-species. Nigam and Kushwaha (1986) made an attempt to distinguish between C. tropicum and C. keratinophilum on the basis of thermo-tolerance of keratinized and non-keratinized propagules. Kushwaha (1983b) highlighted the salient features used in taxonomy of Chrysosporium. Study of scanning electron microscopy (SEM) of Chrysosporium revealed certain features, which were used in its diagnosis, but SEM studies on the

26

morphology of this fungus are rare except Van Oorschot (1980) and Nigam (1987). Recently, the research carried out on Chrysosporium has increased markedly

and

information

has

begun

to

appear.

While

isolating

Chrysosporium by hair-baiting and other methods its potential to degrade

keratin

was

particularly

emphasized.

Perhaps

some

species

of

Chrysosporium may be utilized for recycling of keratin waste in the soil.

Secretion of some of their metabolites, particularly enzymes and antimicrobials is gaining the attention of pharmaceutical

industries. The

resemblance of Chrysosporium to dermatophytes and their pathogenic potential is directly related to the health of human beings and animals. In addition to the keratinous substrate this genus is now being found associated with other non-keratinous substrates too (Kushwaha, 2000). This potential and its similarities to Botryotrichum, Emmonsia, Geomyces, Malbranchea, Myceliophthora, Trichosporiel/a,

Mycogone, Zymonema

ova den dron, and

others

Sepedonium,

along

with

Sporotrichum,

their

associated

teleomorphs is of immense diagnostic importance. Long term studies of Kushwaha (1985, 1987, 1994, 1999), Nigam and Kushwaha (1990b, 1993) on the biology of Chrysosporium, revealed that its wide distribution is due to its antagonistic potential and ability to produce enzymes and other extracellular metabolites. Twenty-two species of Chrysosporium were recognized (Van

Oorschot,

1980) . where

C.

carmichaelii,

C.

crassitunicatum,

C.

evolceanui, C. indicum, C. lucknowensis, c.queenslandicum, C. sulfureum and C. tropicum were isolated from Indian soils (Dominik, 1967; Van

Oorschot, 1980 and Kushwaha, 1985, 1987, 1994). Since then several species have been added to this genus from different habitats around the world. Nigam and Kushwaha (1987, 1989c) isolated some new keratinophilic fungi from Indian soil and house dust respectively. Deshmukh et al. (1985) during a survey of keratinophilic fungi and related dermatophytes isolated C. merdarium from decomposing cow hoof pieces buried in soil samples

collected from cattle farm of Sagar, M.P (India). Kushwaha and Shrivastava (1989) isolated a new species of Chrysoporium from human hairs buried in hospital soil and described it as C. geophilum. Jain et al. (1993) while surveying keratinophilic fungi and related dermatophytes from Indian soils discovered a new keratinophilic species, C. gourii from cattle farm soil of Sagar using defatted human hair as keratin bait. Kaul et al. (1993) isolated 27

three different strains of C. keratinophilum from the soil of dairy farms using feather and horn as baits. Similarly, Singh et al. (1995) observed different strains of Microsporum gypseum from the soil of Agra. Some other new records from different habitats of the world include; C. vallenarense (Van Oorschot and Piontelli, 1985), but the same fungus and

Arthroderma silverae were also isolated from Arctic and Montane habitats by Currah et al. (1996). Chrysosporium europae and C. mephiticum were described by Sigler et al. (1986). AI-Musallam and Tan (1989) isolated C.

zonatum-a coprophilous keratinophilic fungi immersed in horse dung. Skou (1992) worked on a series of xerophilic Chrysosporium species like C.

botryoides, C. farinicola, C. globiferum, C. globiferum variety niveum, C. globiferum variety articulatum C. hispanicum, C. holmii, C. medium, C. medium variety spissescens, C. minor and C. pyriformis. Cano and Guarro (1994) studied the characteristics of C. siglerae. Gene et al. (1994) described C. pilosum, a keratinophilic hyphomycetes isolated from river sediments. Vidal ' et al. (1996) studied a keratinophilic fungi C. vespertilium from Zaire. Chrysosporium fluviale was isolated from river sediments by Vidal et al. (2000), Summerbell (1987) isolated Trichophyton kanei, an anthropophilic dermatophyte. Kane et al. (1992) isolated Trichophyton

krajdenii, an important anthropophilic dermatophyte.

KERATINOLYTIC NATURE OF KERATINOPHILIC FUNGI Evidences of keratinolytic activity on different substrates The common occurrence in nature of microorganisms that readily and in some cases, preferably grow on keratinous substrates has supported the general belief that certain microorganisms can digest keratin. Classical examples of such microbes are the parasitic dermatophytes and the saprophytic onygenales. Keratin is the cornified part of the epidermis of vertebrates and is characterized by having a higher resistance to attack by proteolytic enzymes by virtue of its high cystine content. These cornified appendages include feather, hair, horn, hoof, nail, claw, scale etc. Inspite of the unusual stability and resistance of keratin, feathers and other keratinous wastes do not accumulate in nature, thus evidently confirming the existence of natural degraders. Bird's feathers and animal hairs are the most trouble some waste products and studies concerning their utilization are of great economic and ecological value (Nigam and Kushwaha, 1989a). Physio-chemical methods

28

are mainly used in keratin disposal and processing. These soil inhabiting keratinophilic fungi have special affinity for keratin. The ability of these fungi to invade and parasitize the cornified tissues is ·closely associated with and depends on the utilization of keratin by enzymatic digestion. While observing different keratinous substrates human hair was found to be most resistant. Under

natural

conditions

the

degradation

of

keratin

protein

occurs

microbiologically as a result of the enzymatic and mechanical interaction of keratinophilic

fungi

and

dermatophytes.

Keratinolytic

properties

of

microorganisms provoked interest not only for cognitive but also for practical reasons, hence large number of studies devoted to the methods of identifying these enzymes and their characteristics. Chester and Mathison (1963) and

English (1965) have described the properties related to

degradation of keratin. An index of keratinolytic activity is most often expected as the amount of protein and amino acids or sulphydryl compounds liberated from keratinous substrates compared to controls (Young and Smith, 1975; Deshmukh and Agarwal, 1982, 1985 and Takiuchi

et al., 1984). Thus, the

degree of keratinolysis may be determined indirectly on the basis of the difference in the initial mass of the keratinous material before and after incubating it with a microorganism. Microscopic examinations provide direct evidence of the decomposition of keratin structure, which takes place under the influence of multiplying keratinolytic microorganisms (English, 1965; Kunert and Krajci, 1981; Takatori

et al., 1983; Kaaman and Forslind, 1985

and Bahuguna and Kushwaha, 1989). The first publication dealing with physiological and biological stand-point were published by Stahl

et al. (1949,

1950) to demonstrate how very resistant substrate, keratin is digested and it has been proved that keratinophilic fungi can grow on the hardest keratins as the only source of carbon and nitrogen. The digestion is accompanied by marked alkalization of the medium and by the high activity of proteolytic exo-enzymes in the culture fluid, the cleavage products with the characters of amino acids, peptides and proteins were established. Enzymes with proteolytic and keratinolytic activity were also found in the culture filtrates. Now,

there

are

no

doubts

concerning

the

enzymatic

character

of

keratinolysis in dermatophytes. Various workers (Matsumoto and Agarwal, 1984; EI Naghy

et al., 1983; Takatori et al., 1983; Singh

et al., 1998 and Katiyar and Kushwaha, 1997b, 29

2002) studied the in vitro hair degradation, but feather degradation has received little attention (Nigam and Kushwaha, 1989a; Kaul and Sumbali, 1999b and Parihar and Kushwaha, 1999, 2000). Recently diverse substrates have been shown to become colonized by many keratinophilic fungi and related dermatophytes and are being hydrolyzed intensively and specifically (Mc Donald, 1985; Sanyal et al., 1985 and Kunert and Kasafirek, 1988). Keratin degradation involves rupturing of disulphide bonds between the peptide chains of keratin molecules. Within the group, enzymes are able to break these disulphide bridges of keratin and their presence among taxa should indicate a high level of relationship (Currah, 1985). In order to make some headway towards understanding this clue, attempts have been made by a few workers on the specificity of these enzymes and to discover similarities between the enzymes of different species of keratinophilic fungi and related dermatophytes (Mc Donald, 1985; Kunert and Kasafirek, 1988; Calvo et al., 1991 and Rajak et al., 1991a). The results generally confirmed that shared enzymes and shared catabolic routes both are responsible for the correlated abilities between taxa to use more than one substrate. Kushwaha and Agarwal (1981b) determined the wool degrading capacity of some selected isolates of Aspergillus quercinus, Chrysosporium tropicum,

Microsporum fulvum, M. gypseum, Trichophyton mentagrophytes and T. rubrum. These moulds also showed the utilization of horn keratin. Safranek and Goos (1982) observed degradation of wool by saprotrophic fungi and tested Acremonium roseum and Chaetomium globosum for their ability to attack the protein components of wool by culturing the fungi in basal medium containing wool. Nigam and Kushwaha (1992) observed the course of wool degradation by C. keratinophilum singly or in combination with C.

carmichaelii, C. tropicum and M. gypseum. They studied degradation by measuring the protein released in the culture medium and weight loss up to four weeks. Shrivastava et al. (1996) also observed wool degradation by

Aspergillus niger and Trichophyton simii. The amount of protein released into liquid culture medium and weight loss were taken as the measures of wool degradation. Ghawana et al. (1997) also observed fungal succession during wool decomposition. Singh

and

Agarwal

(1989)

studied

keratinolytic

activity

of 4

keratinophilic fungi: Botryotrichum keratinophilum, Malbranchea aurantiaca,

Nannizia incurvata (+) and N. incurvata (-) strains on various keratin 30

substrates like dear skin, goat skin, camel's hair, buffalo tail hairs, chicken's feather and human nails. Wawrzkiewicz

et al. (1991) tested the ability of 16

strains of 12 species of dermatophytes to utilize keratin substrates (guinea pig hairs and chicken feathers). Rajak

et al. (1992) observed keratinolysis by

Absidia cylindrospora and Rhizomucor pusillus. The keratinolytic activity of these two causal organisms was compared by growing them on a variety of skin appendages of men and animals. Deterioration of feather and leather objects by 5 species of Chrysosporium, 4 of Aspergillus, 2 of Penicillium and 2 each of Acremonium and Fusarium were monitored by Nigam et al. (1994). EI-Naghy et al. (1998) observed feather degradation by Chrysosporium georgiae. Muhsin and Hadi (2001) isolated 4 fungal species including 2 dermatophytes and 2 saprophytes and tested their degradability towards 3 types of keratin substrates (human hair, chicken's feather and wool). Singh

et al. · (1989) discovered a new method just to confirm the

keratinolytic nature of these fungi. To confirm the keratinolytic nature, each individual fungus was inoculated in the middle of the sterile hair, which was tied at each end to a piece of glass rod and kept in a paired petri plate under aseptic condition. A small amount of moist cotton was spread at the bottom of each glass rod to provide humid atmosphere for the growing fungus. This assembly was kept in an incubator for 20-50 days. The fungus developed colony on hair and continued its growth. After a few days, the hair was broken at a point of inoculation indicating the digestion of hair keratin and its disintegration by the test fungi. Time for degradation varied from species to species depending upon its keratinolytic nature. Thus, confirming the keratinolytic nature of the fungus. Hence, it ' was believed that the growth of dermatophytes and keratinophilic fungi on natural keratin substrate and their degradation alone is an evidence of keratinolysis (Kunert, 1972b; Hose and Evans, 1977 and Deshmukh and Agarwal, 1982, 1985). But Weary et al. (1965) gave a view that the intercellular cement of keratin also provides nutrition to microbes. Thus, it can be concluded that these stable and slowly decomposable substrates favour colonization by competitive or combative species (Cooke and Rayner, 1984) in a natural habitat. Griffin (1960) and English (1965) suggested that more physiologically assessable nutrients contained in the keratinous substrates are first utilized, followed by the less assessable nutrients during keratin colonization. 31

Evidences for screening of keratinophilic fungi Screening helped in the selection of strains with exceptionally high keratin degrading ability. It is essential from the point of biotechnology as it helps in the selection of highly significant strains for further experimentation in the field of microbial biotechnology. English (1965) showed that 31 species of saprotrophic fungi were capable of growth on various keratinous substrates. Kushwaha (1983a) studied the degradation of peacock's feather by the enzymes of 20 different fungi and reported that some dermatophytes were active in this respect. Calvo et al. (1985) screened 80 dermatophyte strains

belonging

to

the

Chrysosporium,

genera

Epidermophyton,

Microsporum and Trichophyton for their ability to produce extra-cellular enzymes.

Shrivastava and Kushwaha

(1987) screened

11 isolates of

Microsporum to study hair deterioration. Nigam (1987) and Nigam and Kushwaha (1990b) isolated few highly significant strains of Chrysosporium

carmichaelii, C. keratinophilum, C. tropicum and Microsporum gypseum with high keratin degrading ability during screening for keratinolytic activity. Hasija et al. (1990) tested 30 keratinophilic fungi in vitro to observe their keratinolytic activity to degrade human scalp hair as the sole source of nutrient. Calvo et al. (1991) reported the screening conducted on 390 strains of Chrysosporium to detect their ability to express keratinolytic enzyme activities

in

the

culture

keratinophilic fungi

medium.

for the

Rajak et al.

production

(1991a)

of cysteine,

screened

cystine,

5

inorganic

sulphate, thiosulphate, total keratinase and pH of culture filtrates. Malviya et

al.

(1992a)

examined

26 different species of keratinophilic fungi to

determine their ability to utilize free cystine and observed that the rate of cystine oxidation varied with different fungal strains showing its maximum for Graphium p enicilloide us. Malviya et al. (1992b) also evaluated the keratinolytic potential of Cylindrocarpon lichenicola, Graphium cuneiferum,

M. fulvum and M. gypseum and found that all 4 fungi showed significant extracellular keratinase activity. They also detected inorganic sulphate, thiosulphate, protein, keratinase and change in alkalinity of the culture filtrates during keratin biodegradation of fungi. Lal et al. (1996) screened 55 actinomycetes, isolated for their ability to degrade child's scalp hair keratin as a sole source of nutrient in vitro. Kaul and Sumbali (1999b) compared keratinase activity of 14 species of keratinophilic fungi. They also reported the release of amino acids and proteins during keratin degradation. Muhsin 32

and Salih (2000) screened 16 fungal species, 5 of dermatophytes and 11 of other fungi for the activity of enzyme keratinase, proteinase, lipase and amylase.

Perforatory behaviour of keratinophilic fungi Ajello and Georg (1957) were the first to use the hair perforating activity as a diagnostic tool to see the difference in the ability to perforate hair in vitro among the species of Trichophyton. They showed that this property

could

be

used

to

differentiate

a

typical

isolates

of

T.

mentagrophytes and T. rubrum. All isolates of T. mentagrophytes whether

morphologically typical or atypical, penetrated hair segments forming wedge-shaped perforations. On the other hands, none of the T. rubrum isolate did so. Pinetti and Lostia (1966) in their study on in vitro growth of dermatophytes on hair pOinted out that variation in procedure significantly influenced the mode of hair invasion. In 1980, the in vitro hair perforation test was

used

to characterize 44 species

belonging to the

genera

Epidermophyton, Microsporum and Trichophyton '(Padhye et al., 1980). The

ability to perforate hair in vitro was manifested by 24 of the 44 species, under the original test conditions as stimulated by Ajello and Georg (1957) . The ability or inability to perforate hair was found to be a species-specific character that did not vary among the isolates of a given species. Since sporulating as well as non-sporulating isolates of a given species manifested it, the test was considered as a valuable tool, especially for those species that do not sporulate readily and thus, are difficult to be identified by morphological criteria. Matsumoto et al. (1983) showed 35 isolates of Trichophyton tonsurans variety sulfureum for their ability to perforate hair in vitro using Ajello and Georg (1957) test procedure.

Vanbreuseghem (1952) demonstrated that different dermatophyte species varies in the manner in which they attack hair filaments. The invasion of hair by dermatophytes has been investigated with light and electron

microscopy

under natural

and

experimental

condition.

Light

micrographs showed that naturally infected hair obtained from biopsy specimen were penetrated and channeled by hyphae or arthrospores chain or both (Emmons et al., 1977). The microscopic examination showed extensive hair damage and extensive rupturing of hair cuticle. It was also observed that the keratinophyles differed in their hair hydrolyzing capacities. These results agreed with the findings of Kunert (1972a) and Evan and Hose 33

(1975). It was observed that the level of sulphydryl group was very low throughout the experiment. Yu et al. (1968) had shown that the hair cortex was disassociated in hair fibrils by keratinase(s), however the secretion of enzymes appears to occur at the sites where lamasomes predominated (Kanbe and Tanaka, 1982). Keratinolysis was also demonstrated with the help of light microscopy (English, 1963, 1968 and Raubitschek and Evron, 1963) and electron microscopy (Mercer and Verma, 1963; Poulain and Biguet, 1974 and Hutton et al., 1978). SEM of human hair revealed its biodeterioration

and

longitudinal

furrows

caused

by

Mycelioph th ora

anamorph of Arthroderma tubercula tum. Development of deeper furrows in hair indicated the rapid degradability of the fungus (Nigam and Kushwaha, 1990a). Mercer and Verma (1963); Baxter and Mann (1969) and Kunert and Krajci (1981) also studied human hair deterioration in humid chamber. English (1963) described the stages by which detached hairs are attacked by keratinofers. These are cuticle lifting, cortical erosions, production of penetrated organs and colonization of medulla. Kanbe and Tanaka (1982) also recognized these stages but they overlap one another during the process of infection with Microsporum gypseum. Takatori et al. (1983) observed human hair infected with Microsporum ferrugineum by light and electron microscopy. The fungus invaded the hair

filament and developed hyphae between hair cuticles and cortex. SpherOids of arthro-conidia were frequently observed to invade hair follicles. A mosaic sheeth of massive spheroids were detected around hair filaments. Singh and Agarwal (1984) and Nigam and Kushwaha (1989b) also studied perforation of human hair. 'Cabanes et al. (1987) observed keratinolytic activity of strains of genus Epidermophyton. Shrivastava and Kushwaha (1987) used 11 isolates of Microsporum, out of which 6 were found to perforate hairs in 3 weeks. Bahuguna and Kushwaha (1989) applied in vitro hair perforation test to 8 species of Chrysoporium and 1 species each of Microsporum and Trichophyton and observed different micro-morphological changes caused by

fungal invasion. Nigam and Kushwaha (1990a) studied deterioration of human hair. Cano et al. (1991) studied the human hair destruction by Aphanoascus fulvescens, A. keratinophilus and A. verrucosus. Fusconi and

Marchisio (1991) studied the pattern of invasion of non-dermatophytic fungus C.

tropicum.

Components of hair were attacked in sequence

depending on their level of keratinization. Chrysosporium tropicum thus 34

demonstrated important physiological parallels · with dermatophytes in its way of demolition of hair in vitro. This could be of interest in predicting its ability to infect in vivo. Shrivastava et al. (1993) performed hair perforation test to 7 species of Chrysosporium,

one each of Microsporum and

Trichophyton. Katiyar and Kushwaha (1997a) observed human hair invasion by keratinophilic fungi isolated from the Mediterranean Sea beach. Jahan (1998) studied 15 strains of C. tropicum for colonization and perforation of human hair in soil. Shrivastava and Shrivastava (1999) tested strains of Chrysosporium, Microsporum, Myce/iophthora and Trichophyton for their ability to attack hair filaments. Katiyar and Kushwaha (2000) detected the hair colonizing fungi in water sediments of India. The same workers in 2002 studied invasion and degradation of hair by house dust fungi.

Mode of hair invasion Vanbreuseghem (1952) described the specialized hyphae as 'Organes Perforateurs', which perforate the hair perpendicular to its length, while Page (1950) named them as 'intrusions' and observed partial disappearance of fingernails and

~ow

horn particles. Carmichael (1962) and B6hme and

Zeigler (1967) also remarked that these fungi attack hair by means of 'Penetrating bodies' while Otsenasek and Dvorak (1964) used the word 'Penetrating Organs'. Evolceanui et al. (1963) used the expression 'Organ Perforators' and English (1963, 1969) used the term 'Boring Hyphae' for perforators. Matsumoto et al. (1983) divided two groups of perforators and non-perforators. Perforating group of fungi utilize keratinous substrates present in the hair which results in the damage of hair. During colonization and decomposition of hair various workers reported several types of perforators. Wedge shaped perforators were found in T. mentagrophytes penetrating hair segments (Ajello and Georg, 1957). 5hrivastava (1985) reported torpedo ·shaped perforators in M. fulvum and M. gypseum. Raubitschek (1961) speculated that dermatophytes could disrupt the keratinized tissues purely on mechanical basis without the involvement of chemical keratinolysis. Weary and Canby (1967) also supported this view and found that T. mentagrophytes in spite of lacking chemical keratinolysis caused marked fragmentation and disruption of the wool fibres. English (1965) suggested that breakdown of the characteristic disulphide bonds of keratin molecules is brought about at high pH by the reducing agents. She also discussed about the parallelism between the fronded mycelium and 35

borers of non-keratinophilic fungi with the eroding fronds and perforating organs

of keratinophilic fungi

and

differentiated the

two

groups by

emphasizing that only keratinophilic fungi possess keratinolytic property. English

(1969,

1976) first of all studied the

manner by which the

Chrysosporium species attacked and destroyed human and bovine hair. While observing the destruction of hair by C. keratinophilum, she could not see the true perforating organs as found in dermatophytes. She suggested that the swollen borers of this species are intermediate structures between the narrow borers of non-keratinolytic fungi and the perforating organs of dermatophytes a'nd presumed that the latter might have evolved from the former.

She observed fronded mycelium and terminal and intercalary

appresoria, the thrusts of the fronds may cause cracks in the cuticle but if the penetration is deep a curious undulation of cuticle is seen (English, 1969). Undulation of cuticle may also be noticed in cuticle overlying a frond where there is no visible frond formation but where the cortex below has been strongly attacked. Medulla is next to the cortex. Growth in the medulla is much faster than in the cortex, the keratin of which is easily digested than the trichohyaline of cortex. Medullary rope of mycelium and longitudinal splitting of keratin was also observed. Bahuguna and Kushwaha (1989) observed

cuticle

lifting,

cuticle

disruption,

cuticle

undulation,

narrow

perforating organs, broad perforating organs and complete digestion of hair. According to another view keratins are digested by certain chemical substances or enzymes produced by the dermatophytes. Daniels (1953) determined qualitatively the release of amino acids into the culture medium , during the digestion of hair keratin by Microsporum canis. Weary et al. (1965) determined keratinolytic activity of M. canis and M. gypseum by the release of soluble sulphydryl containing amino acids and polypeptides into the medium in quantities significantly greater than those released by the controls. Morphological studies with electron microscope also suggested a possible induction of enzymes during digestion of keratinized tissues leading to the breakage of disulphide bonds (Mercer and Verma, 1963). Ruffin et al. (1976) discussed that proteolytic activity and sulphitolysis are simultaneous and perhaps complementary phenomenon, exhibited by dermatophytes for transforming keratin into a nutritive material. The proteolytic enzymes, particularly keratinases attracted the attention of dermatologists because of their powerful

action

on

keratin.

Later researches

demonstrated the 36

production of these enzymes by keratinophilic and dermatophytic fungi (Weary et al., 1965; Yu et al., 1971; Takiuchi et al., 1982 and Kushwaha, 1983a). Bahuguna and Kushwaha (1989) categorized various species of Chrysosporium

into

fa rinicola,

keratinophilum,

C.

two

groups

perforating C.

and

lucknowensis,

non-perforating. C.

pannicola,

C. C.

queenslandicum and C. tropicum belonged to perforating group. Nonperforating group of fungi included C. ca rm ich aelii, C. evolceanui and C. indicum. But in the study of Marchisio (1986) and Katiyar and Kushwaha (1997a) all the isolates of C. indicum were found to be positive. This may be considered as a possible cause of the keratinophilic fungi isolated from the different habitats i.e. Sandpits of Turin (Marchisio, 1986), Mediterranean Sea beach of Spain (Katiyar and Kushwaha, 1997a). The variation in the degree of perforation among the different isolates of the same species could represent their taxonomic significance and can be recognized as a subvariety (Matsumoto et al., 1983). Fusconi and Marchisio (1991) observed the demolition of hair in vitro by C. tropicum. He found that fungus attack on hair by two ways, one mode of attack depends upon the level of keratinization and in another mode the attack was unaffected of degree of keratinization.

EVIDENCES OF GROWTH OF KERATINOPHILIC FUNGI ON DIFFERENT NUTRITIONAL MEDIUMS. Various workers have proved the versatility of the nutritional range of these fungi. Reports on the nutritional studies of keratinophilic fungi involved those of Kushwaha and Agarwal (1975, 1977 and 1981a) and Kushwaha (1976, 1984). Singh

and

Agarwal

(1982)

studied

spore

germination

of

Chrysosporium crassitunicatum, Nannizia fulva (+) and (-) strains and Trichophyton equinum in the presence of various carbon and nitrogen sources.

The

nutritional

requirement

including

carbon

sources

were

arabinose, glucose, lactose, maltose, mannose and starch and the nitrogen sources were alanine, aspartic acid, cystine, glutamic acid and proline. Maximum spore germination within 24 hours was recorded when glucose was used as a carbon source for all the test fungi. Among the inorganic nitrogen · sources were ammonium nitrate, ammonium sulphate, sodium nitrate. Except sodium nitrate all inorganic nitrogen sources enhanced spore 37

germination at 0.05% concentration. Most of the organic nitrogen sources used were found to be stimulatory for the spore germination of the test fungi. Singh (1983) studied radial growth of C. crassitunicatum, N. fulva (+) and (-) strains and T. equinum on 10 different mediums- tryptone agar, YPSS agar, SDA, oat meal agar, malt extract agar, synthetic acid agar, PDA, Czapeck's dox agar, glucose asparagine agar and Czapeck's dox agar + yeast extract. It was revealed that SDA among the natural media and glucose asparagine agar among synthetic media were best suitable for the growth of selected test fungi. Singh (1984) studied vitamin requirement of keratinophilic fungi and related dermatophytes. Basak and Samajpati (1986) studied the effect of di-alpha tocopheryl acetate, vitamin A, ascorbic acid and riboflavine on the growth of two dermatophytes Aspergillus fumigatus and T. mentagrophytes. Singh et al. (1995) isolated a variety of strains of M. gypseum and observed the effect of different culture media (glucose

asparagine medium, glucose gelatin medium, PDA, Richard's medium and SDA) in order to find out their nutritional requirement and found that SDA was preferred most, followed by glucose gelatin medium, PDA, Richard's medium and glucose asparagine medium. Malviya et al. (1993c) studied sulphur utilization by Scopulariopsis brevicaulis using sugar and amino acids as carbon sources. Maximum

production of inorganic sulphate was observed in the growth medium containing sucrose as a main source of carbon. Amino acids supported comparatively lower oxidation of sulphur. Highest mycelial biomass was obtained when cultured in a medium containing galactose as a main source of carbon. Many reports are there about carbon rich media as the growth media of keratinophilic fungi, but are of questionable relevance to our understanding of fungal physiology (Kunert, 1973 and Sanyal et al., 1985). Some authors have studied fungal sulphur oxidation in the medium containing relatively low concentration of sugars. The ability of fungi to grow in low carbon medium (oligotrophy) plays an important role in the growth of these organisms in the soil. This ability may help fungi to oxidize sulphur in keratin rich habitats like gelatin factory site (Rajak et al., 1991a). Natural keratin contains 2.5% sulphur as a part of it (Kunert, 1973). Nigam

and

Kushwaha

(1988)

studied

morphological

growth

characterisitics of many species of Chrysosporium and their growth pattern on different medias. Fifteen species of Chrysosporium were grown on 38

following medias- gelatin agar, keratin agar, malt extract agar, mineral agar, Sabouraud's dextrose agar (SDA), starch agar and soil extract agar. The growth was measured as a colony diameter. Besides this group of fungi, different nutritional studies on other fungal isolates include those of Casal and Linares (1981); Rai (1989); Coty (1994) and Llop

et al. (1999).

INTERACTION

BETWEEN

PATHOGENIC

AND

SAPROPHYTIC

KERATINOPHYLES Griffin (19'6 0) demonstrated that initial colonizers of hair were fungi with high saprophytic ability; they gave way to less competitive fungi, which in turn were usually replaced by keratinophilic fungi. Wicklow (1981) suggested that fungi of antagonistic activity, which would involve the production of anti-microbial agents or direct hyphal interference, colonized pre-colonized substrates. Mycoparasitism and hypha I interference between several taxonomic groups of fungal species determine in vivo and in vitro the pattern of colonization (Ikediugwu and Webster, 1970a, b; Rayner and Todd, 1979; Shafie and Webster, 1979; Arora, 1980; Arora and Dwivedi, 1980 and Upadhyay

et al., 1981). Shearer and Zare-Maivan (1988) studied hypha I

interference in an aquatic environment.

However, scanty reports are

available' on the study of hyphal interference and antagonism among keratinophilic fungi (Nigam and Kushwaha, 1990c; Ghawana, 1997 and Shrivastava

et al., 2002).

Phenomenon of stimulation is rarely reported but they are commoner than is presently realized, it has been described by Morton and Eggins (1976). Often hyphae of different species intermingle in culture with no perceived benefit or disadvantage to either mycelium, but if some degree of mutual intolerance exists, opposing mycelia will eventually stop growing, even though this may be after contact between the two mycelial has been established, such mycelia remain visible and distinct forming discrete units. They may respond on contact by constructing defensive hyphal barriers at mycelial fronds to limit the advancement of the aerial mycelium. These restrictions on growth arise as a result of mycelial response to the release of metabolic inhibitors like antibiotics, enzymes or to limitation imposed on growth by competition for nutrient, oxygen or even space. These are referred to as indirect antagonism since they don't require hypha I contact to take effect, but act in advance of the extending mycelium in the more 39

aggressive forms of behaviour, the hyphae of one competitor will advance into the mycelium of the other and be destroyed by overgrowth and through hyphae to hyphae interaction. These are known as direct antagonism since they rely on contact and cover such phenomenon as mycoparasitism and hypha I interference. Indirect antagonism involves, antibiosis and competition for substrate and space. If one fungus is more successful ·in competing for available substrate, this will prevent supplies reaching to others and thus, will create an antagonism or harmful effect. Many fungi are known to produce fungicidal metabolites, which diffuse from hyphae and slow down or stop the growth of competitors from some distance. If two opposing species produce inhibitory metabolites mutual inhibition may result. Inhibition by antibiosis is often species-specific and response occurs only when appropriate species meet. Antibiosis phenomenon is associated with capture of resource and with the defense of already secured

resources from take over by would

be

competitors. Antibiotics produced by fungi consist of both volatile and nonvolatile substances. These may be complex secondary metabolites or some simple products of primary metabolism such as alcohols and aldehydes. The latter are auto-toxic and their accumulation in culture media can lead to the arrest of growth, disruption of metabolic pathways, breakdown of organelles and lysis. Reviews of Hutchinson (1971) and Fries (1973) contain a list of some volatile antibiotics of different kinds, which are commonly produced by fungi of all the . taxonomic groups, some of which have been identified. Among these compounds aldehydes, ammonia, ethylene, ethyl alcohol and hexa-1,3,S

trysine

are

known

to

be

inhibitory

to

fungi

at

certain

concentrations. Dennis and Webster (1971a, b) had investigated many antifungal volatiles and some have been identified (Clayton et al., 1987). Dennis and Webster (1971a, b) have shown that acetaldehyde can be inhibitory at concentration as low as lSppm. Trichoderma species are particularly active producers

of several

anti-fungal

volatiles.

Non-volatile antibiotics are

produced by many fungi and cover a whole range of chemically diverse secondary metabolites. Soil fungi in the genera Aspergillus, Penicillium and Trichoderma are very profilic producers. Citrinin, patulin, glitoxin and

penicellic are some of these produced by Aspergillus and Penicillium species. Acremonium species are also known to produce antibiotics (Kazma et al.,

1993). 40

In vitro studies of Nigam and Kushwaha (1990c) revealed that Chrysosporium tropicum was inhibited by staling substances of Aspergillus flavus, A. niger, Chaetomium g/obosum, Chrysosporium evo/ceanui, C. indicum, C. /ucknowensis, C/adospora species, M. gypseum, 3 species of Penicillium, and T. vanbreuseghemii. The ability of C. tropicum to interact

with 12 keratinophilic fungi and saprophytic fungi was evaluated in dual cultures. Frequent curling, penetration, granulation, lysis and chlamydospore formation in C. tropicum were observed during hyphal interference. The effect of fungal staling substances of 8 Chrysosporium species on soil mycoflora was studied by 8ahuguna and Kushwaha (1992) and it was found that among 4 strains of C. tropicum, one allowed a minimum number of fungi to grow, staling substances of other Chrysosporium species also caused inhibition of soil fungi. Metabolites secreted by C. evo/ceanui, C. pannico/a, and C. queens/andicum showed strong anti-fungal activity against A. niger (8ahuguna and Kushwaha, 1992). Saxena and Kushwaha (1992) also carried out interaction experiments on C. anamorph of Arthroderma curreyi. The inhibition and promotion in dual cultures depends on many factors such as staling products of interacting colonies, pH change, nutrient media depletion

or

alternation

of

nutritional

ingredients

besides

hypha I

interference. Colony interaction in a number of cases is represented by mutual inhibition in the growth of both fungal partners. The relative difference reveals the measure of susceptibility and antagonistic ability of the fungus. The development and subsequent formation of chlamydospores, lysis, coiling, deformation, granulation and swelling in dual cultures in most cases is an evidence of their successful competition (Nigam, 1993).

ANTI-FUNGAL ACTIVITIES OF PLANT EXTRACTS. Infectious diseases are an important health hazard allover the world. A superficial ringworm infection is more frequent than subcutaneous or systemic mycoses and remains a therapeutic problem in tropical and subtropical countries (Stern, 1996), despite the availability of a number of antifungal ointments, paints, lotions and powders. Sources of these agents are largely non-renewable petro-products that are not biodegradable and cause adverse effects and residual toxicity (Shahi et a/., 1999). Though new antibiotics are continuously emerging in the market because of their effectiveness against harmful microbes but it's over use has applied a question mark on their future effectiveness. The

main drawback of using 41

these drugs is that they act upon the system as foreign bodies depressing and

paralyzing

its

function

due to their

persistent and

consequent

accumulation. So, these need to be supplemented with other anti microbial agents. Plants oil and extracts have been used for a wide variety of purposes for many 1000 of years (Jones, 1996). In particular, the anti-microbial activity of plant oils and extracts has formed the basis of many applications including pharmaceutical alternative medicines and natural therapies (LisBalchin and Deans, 1997). Higher plants are untapped reservoirs of various chemical.s awaittng intensive exploitation for their biological properties. Hence, now days, a large number of anti-microbial agents have been produced to combat a wide variety of microbial and fungal infections. Recently, some products of higher plant origin have been shown to be an effective source of chemotherapeutic agents and provide renewable sources of useful anti-fungals of biodegradable nature, which are devoid of any side effects. Besides, they are effective as fungitoxicants due to low phytotoxicity, more systemicity, easy biodegradability and stimulation in host metabolism. These plants derived compounds (Phyto-chemicals) have been attracting much interest as natural alternatives to synthetic compounds. The medicinal value of plants is due to the presence of certain secondary metabolites

such

as

alkaloids,

f1avonoids,

saponins,

gums,

terpenes,

glycosides, resins, tannins, volatile oils, aliphatic acids and aldehydes etc. Over the last 40 years intensive efforts have been made to discover clinically useful anti-fungal drugs (Valsarag et al., 1996; Werner et al., 1999 and Perumal Samy and Ignacimuthu, 2000). The increasing interest on traditional ethno-medicine may lead to the discovery of novel therapeutic agents. Natural products of higher plants may offer a new source of antimicrobial agents for external use like gargles and ointments (Brantner and Edith Grein, 1994). These findings prompted the exploration of various plant products that could be exploited as anti-microbials. A perusal of literature documented that many investigators have reported anti-bacterial (Mehta et

al., 1983; Hammer et al., 1999; Sandhu and Arora, 2000; Bhadauria et al., 2001; Hazra et al., 2003; Shashikumar et al., 2003 and Sheela and Kannan, 2003) and anti-fungal (Bilgrami et al., 1980; Kumar and Kumar, 1980; Mishra and Dixit, 1980; Singh et al., 1980; Bhowmick et al., 1981; Charya and Reddy, 1981; Natarajan and Lalithakumari, 1987; Mishra et al., 1989; Gaurinath and Manoharachary, 1990; Tariq and Magee, 1990; Upadhyay and

42

Gupta, 1990; Kishore and Mishra, 1991, Ranjan et al., 1991; Kumar and Chauhan, 1992; Kumari and Jariwala, 1993; Gohil and Vala, 1996; Narayan Rao et al., 1996; Rai, 1996; Purohit and Bohra, 1998; Muhsin et al., 2000; Bajpai and Singh, 2003 and Suhr and Neilsen, 2003) properties of extracts of higher plants. As has already been discussed that dermatophytes have been reported to be potentially pathogenic (AIi-Shtayeh and Arda, 1985) and are directly connected with the skin fungal infections, there are various reports exploring

the

medicinal

plants

and

their

inhibitory

nature

against

dermatophytic fungus. Banerjee et al. (1982) studied the inhibitory effect of oil .of Curcuma amada against M. gypseum and T. mentagrophytes and other saprophytic fungi. Garg et al. (1985) observed inhibition in the growth of

Ctenomyces serratus, M. gypseum and T. ajelloi by a variety of oil formulations. Singh et al. (1986) evaluated the anti-fungal properties of essential oil of Ageratum conyzoides against E. floccosum, M. canis and T.

mentagrophytes. Dube and Tripathi (1987) screened 6 plants for their antifungal activity against E. floccosum, M. canis and T. mentagrophytes. Mares (1987) tested proto-anemonin, a compound obtained from Ranunculus

bulbosus as an anti-fungal agent on some strains of dermatophytes and yeast and found that E. floccosum was the most sensitive dermatophyte against it. Mustard and linseed oil were found to be most inhibitory in the study performed by Nigam et al., (1987). Rai and Upadhyay (1988b) also observed the effect of oil of Mentha piperita against T. mentagrophytes. Singh and Agarwal (1988) used the essential oils of 7 medicinal plants against

the

mycelial

growth

of 4

keratinophilic

fungi

Botryotrichum

keratinophilum, Malbranchea aurantiaca, Nannizia incurvata strain (+) and strain (-). Dixit et al. (1990) screened essential oils of different plants for their

toxicity

against

ringworm

fungi

belonging

to

the

genera

Epidermophyton, Microsporum and Trichophyton. ryer and Williamson (1991) studied 3 species of Trichophyton (T. mentagrophytes, T. rubrum and T.

violaceum) to detect the protease but 100% extracts of Allium sativum, Ocimum sanctum,

Catharanthus roseus and Azadirachta indica totally

inhibited the protease activity of these fungi. This protease is considered as an important weapon of the dermatophytes for the establishment of functional host paraSite relationship. Shrivastava et al. (1997) studied the efficacy of Artemisia extract on hair perforation activity of M. nanum and T. 43

rubrum.

Plant extracts of Lawsonia inermis,

Eclipta alba, Nyctanthes

arbortristic, Datura stramonium and a mixture of all the extracts were used for testing the anti-fungal activity of C. tropicum (Singh and Singh, 1997). Essential oils of Mentha arvensis, Trachyspermum ammi, Cymbopogan

nardus and Eucalyptus citridora also caused more than 77% inhibition of C. tropicum (Singh et al., 1997). Rai and Upadhyay (1988a) screened 19 medicinal plants against T. mentagrophytes. Lachoria et al. (1999) screened foliar part of 26 plants for their anti-fungal activity against 4 strains of dermatophytes (E.

floccosum,

Microsporum sp., M.

gypseum,

and T.

rubrum). Shahi et al. (1999) evaluated the anti-fungal nature of essential oil of some Eucalyptus spp. against dermatophytes E. floccosum, M. gypseum,

M. nanum, T. mentagrophytes, T. rubrum and T. violaceum. Vaijayanthimala et al. (2001) reported the anti-fungal activity of oils of Arachis hypogea, Coccus nucifera, Olea europaea, Pongamia pinnata, Ricinus communis and Sesamum indicum on T. rubrum and T. mentagrophytes. Agarwal et al. (2004a, b) extracted aerial parts of Phyllanthus amarus and Boerhavia

diffusa observed anti-fungal activity against M. gypseum. Hence, it seems that in the present year, there has been a gradual revival of interest in the use of medicinal plants in developed as well as in developing countries due to high cost and harmful side effects of antibiotics. So, the need of the day is to explore more and more potential metabolites of plant origin that can mimic the effect of present drugs and supplement with other more effective drugs that are more easily available to mankind.

ENZYMATIC ANALYSIS (MICROBIAL KERATINOLYSIS)

Biochemical evidence of keratin degradation The ability, of these fungi to invade and parasitize cornified tissues is closely associated with the utilization of keratin by enzymatic digestion. The soil

inhabiting

keratinophilic

fungi

Aphanoascus,

Arthroderma,

Chrysosporium, Ctenomyces, Malbranchea and Nannizia share this capacity with dermatophytes. When the pathogenic strains, surviving in soil finds a suitable host in favourable environment and physiological conditions they produce the symptoms of ringworm disease. Thus, the virulence factor is of considerable importance and keratinase produced by this fungus is the major enzyme involved with this pathogenesis process or virulence factor (Howard, 1983).

Evidence

is also available on

correlation

between

proteinase

(enzyme) activity of isolates of some fungi and their virulence in vivo or in

44

animals (Asahi et al., 1987 and Lovie et al., 1994). Keeping the above view in

mind,

it was

thought desirable to

work

on

the

mechanism

of

biodegradation of keratin. Under natural conditions these keratinolytic microbes digest keratin by means of keratinolytic system that includes active alkalization of the substrates, extracellular sulphitolysis of disulphide bonds and the proteolysis of keratin molecules. Degradation of keratinous material has been studied by various workers ' (Kunert, 1972a, 1973, 1976a; Evan and Hose, 1975; Kushwaha and Agarwal, 1981b; Safranek and Goos, 1982; Wainwright, 1982; Deshmukh and Agarwal, 1982, 1985; Kushwaha, 1983a, 1998; Wawrzkiewicz et al., 1987; Nigam and Kushwaha, 1989a, 1990a, d, 1992; Rajak et al., 1991a, 1992; Malviya et al., 1991, 1992a, b; Singh et al., 1996 and EI Naghy et al., 1998) and it was proved that-the keratinophilic fungi can grow even on hard keratin as an only source of carbon and nitrogen. The degradation of keratin in mineral medium is accompanied by alkalization of the medium . Moreover,

Lal

et

al.

(1996)

studied

keratin

degradation

by

actinomycetes. Noval and Nickerson (1959); Nickerson et al. (1963); Young and Smith (1975) and Sinha et al. (1991) showed the keratinolytic activity of actinomycetes. It was demonstrated that among several hundred's strains of

Streptomycetes the highest proteolytic activity was shown by S. fradiae and enzymatic activity was manifested by both actively growing microorganism (Noval and Nickerson, 1959) and cell free filtrates (Young and Smith, 1975). Similarly, Friedrich and Antranikien (1996) studied keratin degradation by

Fervidobacterium pennavorans. Suntornsuk and Suntornsuk (2003) observed feather degradation by Bacillus sp. FK46 in submerged cultivation. Non-pathogenic fast growing keratin degrading fungi have been found implicated in keratin digestion in recent days. Non-keratinophilic fungi were also found implicated in keratin decomposition (Kunert, 1995). Reports on their frequent occurrence were made by Evan and Hose (1975); Safranek and Goos (1982); Nigam and Kushwaha (1989d); Nigam and Kushwaha (1992); Kushwaha (1995) and Kushwaha and Nigam (1996) gave the biochemical evidence supporting observations that non-dermatophytic fungi can attack and utilize keratinous substrates. Singh and Agarwal (1987); Nigam

and

Kushwaha

(1992)

and

Kushwaha

(1995)

studied

keratin

degradation by soil inhabiting fungi. Deshmukh and Agarwal (1982, 1985) 45

studied degradation of human hair. Singh and Agarwal (1989) studied keratin utilization by some keratinophilic fungi. Hasija et al. (1990) also studied keratinolytic potential of some fungi. Malviya et al. (1991) made preliminary study on the keratin degradation by Scopulariopsis brevicaulis isolated from wool. Nigam and Kushwaha (1992) studied wool degradation by C. keratinophilum singly and in combination with other fungi. Rajak et al. (1991a, 1992) studied keratin degradation and also compared keratinolysis caused by Absidia cylindrospora and Rhizomucor pusillus. Singh and Singh (1995) and Deshmukh and Agarwal (1998) monitored the role of C. tropicum in keratin degradation. Singh and Singh (1995) also studied characterization of extracellular proteolytic enzyme of C. tropicum. It also degraded buffalo horn, women's hair and wool. Shrivastava et al. (1996) studied wool degradation

by Aspergillus niger and Trichophyton simii.

Parihar and

Kushwaha (2000) tested 100 keratinophilic fungi belonging to Acremonium,

Aphanoascus, Botryotrichum, Chrysosporium, Ctenomyces, Gymnoascus, Malbranchea, Microsporum, Narashimella and Verticillium for their potential to utilize hen's feather as keratin waste. Muhsin and Hadi (2001) studied keratin degradation by fungi isolated from sewage sludge. Dodia and Singh (2002) studied in vitro keratin biodegradation on certain keratin substrates by keratinophilic fungi isolated from patients of ringworm infection.

Keratinase production: a biochemical proof It · is now well established that keratinolysis is due to enzyme action

(Mercer and Verma, 1963 and Singh and Agarwal, 1987) and some intracellular and extracellular enzymes collectively called keratinases carry out

destruction

of

keratin

by

fungi.

Isolation

of

keratinases

from

dermatophytes and their activity has been well established (Weary et al., 1965; Yu et al., 1971 and Kushwaha, 1983a). Now at present there is no doubt that keratinolysis in fungi is an enzymatic process, although a certain role of mechanical substrate breakdown cannot be excluded. Enzymatic lysis is also indicated by the measurable weight loss of the substrate even in the hard keratin degraded by the fungi in vitro (Kushwaha and Agarwal, 1981b; Deshmukh and Agarwal, 1982, 1985 and Kunert, 1989a) liberation of large amount of solubie products (especially peptides and amino acids) into the medium (Deshmukh and Agarwal, 1982, 1985; Safranek and Goos, 1982; Kushwaha, 1983a; Kunert, 1989a, b; Hasija et al., 1990; Malviya et al., 1991, 1992b and Rajak et al., 1991a, 1992) or conversion of nitrogen and 46

sulphur originally contained in protein, into inorganic compounds (ammonia, sulphate) excreted into the medium. Histo-chemical methods also revealed important chemical changes of the substrates in the vicinity of penetrating fungal hyphae (Kamalam and Thambiah, 1981 and Safranek and Goos, 1982). A significant role in keratin degradation is played by extracellular proteases. These enzymes are most active in cultures where keratin is the main or only source of nutrition. The attack of proteases of dermatophytes on keratin is faster and more efficient than that of non-specific proteases like trypsin or papain (Stahl et al., 1949, 1950; Chattaway et al., 1963; Zeigler, 1966; Yu et al., 1968, and Kunert, 1976b). Finally the immuno chemical proof of the excretion of proteases from the hyphae penetrating the keratinized tissues in vivo (Grappel and Blank, 1972 and Lee et al., 1988, 1990) and in vitro (Sei . et al., 1982) indicates the involvement of these enzymes in keratin degradation. Therefore, proteases of keratinophilic fungi are often termed as keratinases. Keratinase is an enzyme inducible by the addition of keratinic sources as substrate in low amount to a peptone medium (Takiuchi et al., 1982). On the other hand, many data suggest that the effect of enzymes of keratinolytic fungi on native keratin is rather limited. The release of soluble products from keratin decreases with time and may even cease (Chester and Mathison, 1963; Yu et al., 1972; Kunert, 1976b and Takiuchi et al., 1982, 1984). Various workers have isolated extracellular keratinases from T. mentagrophytes (Yu et al., 1968), M. gypseum (Takiuchi and Higuchi, 1977), M. canis (Takiuchi et al., 1982) and reported that keratinase are able to digest keratin fibrils. The process of degradation of keratin is a result of both mechanical action of the fungus (Raubitscek, 1961 and Baxter and Mann, 1969) and the enzymatic proteolytic activity of intracellular keratinases (Yu et ai, 1971 and Sanyal et al., 1985) connected with cell walls (Wawrzkiewicz et al., 1987) or released to the medium (Kushwaha, 1983a; Takiuchi et al., 1984 and Sanyal et al., 1985). Kushwaha and Nigam (1996) monitored keratinase production by some geophilic fungi. Kaul and Sumbali (1999b) studied extracellular keratinase production by keratinophilic fungal species . Singh (1999) studied exocellular proteases of Malbranchea gypsea and their role in keratin degradation. Singh and Singh (1995) isolated extracellular proteolytic enzymes of C. tropicum and observed its role in keratin degradation. Yu et al (1971); Higuchi and Takiuchi (1980); Takiuchi et al.

47

(1984); Sanyal et al. (1985) and Singh and Agarwal (1989) purified, crystallized and studied the physio-chemical properties of proteases of several dermatophytes and related keratinophilic fungi. These workers also emphasized on its role in keratin degradation and mycotic infections in host tissues. Muhsin and Aubaid (2000) purified exocellular keratinase from T.

mentagrophytes variety erinacei. Moreover, different Trichophyton species were reported as exocellular enzyme producers (Apodacca and Mackerrow, 1990; Brasch and Zaldua, 1994; Samadani et al., 1995 and Ibrahim et al., 1996). Bockle et al. (1995) also isolated keratinolytic serine proteinase from

Streptomyces pactum. Keratinase purification and characterization has also been studied from feather degrading Bacillus licheniformis strain (Lin et al., 1992) from Bacillus sp. AH-101 (Takami et al., 1990), from S. fradiae (Sinha

et al., 1991). Okafar et al. (1996) observed extracellular enzyme activity of Histoplasma capsula tum variety duboisii. Oyeka and Gugnani (1995) isolated extracellular protease of Hendersonula torruloidea, Scytalidium hyalinum and S. japonicum . Zhu et al.

Aspergillus

flavus

and

(1990) detected extracellular proteases of

studied

its

relation · in

fungal

keratitis

and

pathogenesis. Malviya et al. (1992c) conducted a study to purify and partially characterize

the

extracellular

keratinases

of

fungus

Scopulariopsis

brevicaulis. These studies on different fungal isolates showed considerable variation in their ability to hydrolyze keratin (Rajak et al., 1991a, band Malviya et al., 1992c). Singh and Agarwal (1987) studied protease production by certain keratinophilic fungi. Calvo et al. (1985) studied extra-cellular enzymatic activity of dermatophytes. Yu et al.

(1971)

extracted

2 cell

bound

keratinases from mycelium of T. mentagrophytes. Malviya et al. (1992d) compared

keratinase

induction

activity

and

repression

in

between

ChrysosfJorium queenslandicum, Graphium penicilloideus and Scopulariopsis brevicaulis. Muhsin and Salih (2000) observed exocellular enzyme activity of dermatophytes and other fungi. He not only observed keratinase activity but also detected amylase, lipase and proteinase activity of these fungi. Asahi et

al. (1987) purified and characterized extracellular proteinases from T. rubrum-an anthropophilic fungus, which use human skin protein as a nutrient source. 48

Thus, it has been seen that specific protease released intracellularly or extracellularly

by

keratin

degrading

microorganisms

are

keratinolytic

enzymes. These enzymes have the capacity to act on compact substrates better

than

distinguishes

other

comparable

keratinase

from

proteolytic

other

enzymes;

proteases

and

this

property

peptidases.

Many

keratinases are active extracellularly, being exported from the intracellular sites of synthesis. However, evidence for cell-associated activity has been reported from proteinase of T. mentagrophytes (Yu et al., 1971) and T. rubrum, a well-studied keratin-degrading microorganism (Lamkin et al.,

1996). However"the endo-protease of T. rubrum exhibited a broad spectrum for

protein

substrate,

unlike

that

of T.

gallinae.

Varying

microbial

keratinolytic activities and enzymatic substrate-specificity may be due to the methodology used and species differences. For instance, Lin et al. (1992) found that keratinase from Bacillus licheniformis was capable of hydrolyzing all protein substrates tested, including bovine serum, albumin, collagen and feather keratin. Consistent with the above, Bockle et al. (1995) observed liberation of peptides from different soluble substrates (casein, gelatin) and insoluble substrates (native and autoclaved chicken's feather). On the other hand, Dozie et al. (1994) reported a thermophilic keratinolytic proteinase from C. keratin ophilum, which hydrolyzed only keratin but showed no activity on casein, bovine serum, and albumin. This indicates that the isolated enzymes were substrate-specific. These enzymes have usually been found associated with the mycotic infections of men and animals. Grappel and Blank (1972) demonstrated the role of keratinase in hypersensitivity reaction. Das and Banerjee (1982) correlated the failure of experimental infection in guinea pigs by T. rubrum udar mutant, with its inability to secret enzyme keratinase in vitro. Thus, it is obvious that the keratinophilic fungi, specially the dermatophytes possess specific enzymes that enable their parasitic growth on highly resistant keratinous substrates and hence, all the evidences suggest the role of keratinases in keratin digestion.

Mechanism of microbial keratinolysis Keratinophilic fungi when cultured in medium containing keratin may degrade and use this compound as a basic source of carbon and nitrogen. However, the way in which these fungi attack and digest keratin is not yet thoroughly

known.

Mechanical

penetration

followed

by

enzymatic 49

degradation

has

been

suggested.

Mechanical

penetration

of

keratin

degrading fungi and its mycelium has already been discussed before, but the enzymatic degradation or microbial keratinolysis depends on the secretion of extracellular enzymes, which have the ability to act on compact substrates. The reason for the mechanical stability of keratin and its resistance depends on the tight packing of the protein chain in a-helix or l3-sheet structures. Keratin is an insoluble fibrous proteinaceous, complex filling the terminally differentiated cells of the epidermis. These protein chains are composed of long polypeptide chains joined together by peptide linkages and each amino acid in a peptide is connected to adjacent amino acid by intra-hydrogen bonds. The long axes of peptides are oriented along the length of keratin fibrils in a spiral configuration as revealed by electron microscopy (Mercer and Verma, 1963). It is now well established that keratin stimulates the development of keratinophilic fungi and dermat6phytes and increase their distribution. Keratin release is possibly carried out by extracellular enzyme, keratinase produced by this unique group of fungi. The mechanism by which keratinophilic fungi degrade keratin is quite disputable. Kunert (1973) reported a release of cysteine, cystine and sulphate in the culture filtrates of M. gypseum growing on hair. Ruffin et al. (1976) detected the presence of S-sulphocysteine in the culture fluid and confirmed

the

role

of

sulphitolysis

during

keratin

degradation

by

Keratinomyces ajelloi. However, Stahl et al. (1949); Chester and Mathison

(1963) and Ziegler and B6hme (1963) could not detect cysteine or thiols in the culture filtrates of the dermatophytes they studied. Weary and Canby (1967) recorded the production of 21 and 38 !-Ig/ml of cysteine by 2 strains of T. rubrum . These findings were of special interest and suggested that there is an immediate need to analyze the role of sulphitolysis during keratin biodegradation. However, several other workers emphasized on the manner in which

keratinophilic dermatophytes attack and digest keratin

rich

substrates and said that degradation is proteolytic possibly with keratinolytic enzymes.

Deshmukh and Agarwal (1982, 1985) studied the in vitro

degradation of human hair by various soil inhabiting keratinophilic fungi and observed that breakdown of hair is characterized by the release of protein, peptide and amino acids and also resulted in marked ·alkalization of the medium.

DEI Central Library 111111111111 111111111111 1111 TH208

50

However, Kunert (1988a) stated that sulphur metabolism (cysteine and cystine) plays a significant role in the physiology of human pathogenic fungi and dermatophytes. These fungi can utilize keratin, a sclero-protein with a high sulphur or cystine content. They can utilize cystine as a sulphur source and in addition as a source of carbon and nitrogen and thus, actively metabolize cystine added to the nutrient media. Thus, eliminate the relative excess of sulphur in this compound (26.7%) by oxidation and excretion to the medium mainly in the form of sulphate (Danew et al., 1981, 1982; Kunert 1972a, c, 1975, 1982, 1985a, b, c). It was found that dermatophytes also produce sulphite that does not remain free but reacts with the residual cystine in the medium to yield cysteine and S-sulphocysteine according to:

Cys

I

+

sol+

~

~ Cys + CYS-S03- (so called sulphitolysis)

Cys As this reaction also proceeds in protein bound cystine, a hypothesis on the key role of sulphiotolysis in the decomposition of keratin by fungi has been postulated (Kunert, 1972b). According to this hypothesis the excreted sulphite reacts with cystine bridges of keratin, which is denatured by cleavage

of disulphide

bonds and

can

be subsequently attacked

by

proteolytic enzymes of the fungus. Cystine catabolism and its role in keratin decomposition were then studied in a series of papers (Kunert, 1973, 1975, 1976a, 1982, 1985a, b). It has been confirmed that oxidation of cystine to sulphate is a key reaction in keratinolysis (Stahl et al., 1949; Kunert, 1973, 1989a and Danew et al., 1982) . Keratin denatured in this way becomes easily accessible to proteolytic enzymes of fungi (Ruffin et al., 1976). Hence,

the

studies

on

the

biochemical

mechanism

of keratin

degradation led to the conclusion t hat proteolytic cleavage of keratin protein occurs only after denaturation of the substrate. The fungi accomplish it by sulphitolysis of disulphide bridges, which are the main source of extra ordinary keratin resistance. The mycelia utilize cysteine from the keratinized tissues as the source of carbon and nitrogen and excrete excess sulphur, after oxidation, back into the medium as inorganic sulphate and sulpite. The former compound is a true excretory product while sulphite is used for cleavage of the disulphide bonds of the substrates. Keratin denatured in this way is than attacked by extracellular proteases of the fungus (Kunert, 1972a, c; 1973; 1976b). The authors found an accumulation of sulphydryl 51

compounds (probably cysteine containing peptides) in the medium and presented a hypothesis on the denaturation of substrate by direct reduction by means of enzymes on the mycelial surface. At present there is no doubt that keratinolysis in fungi is an enzymatic process although a certain role of mechanical substrate breakdown cannot be excluded. Enzymatic lysis is also indicated by the measurable weight loss of the substrate even in the hard keratin degraded by fungi in vitro (Kushwaha and Agarwal, 1981b; Deshmukh and Agarwal, 1982, 1985 and Kunert, 1989a) ,liberation of large amount of soluble products (especially peptides and amino acids) into the medium (Deshmukh and Agarwal, 1982, 1985; Safranek and Goos, 1982; Kushwaha, 1983a; Kunert, 1989a, b; Hasija et a/., 1990; Malviya et a/., 1991, 1992b and Rajak et a/., 1991a, b, 1992) or conversion of nitrogen and sulphur originally contained in protein into inorganic compounds (ammonia and sulphate) excreted into the medium. It leads to an assumption that the fungi are also able to denature keratin prior to an attack by proteases. By analogy with other keratinolytic organisms, this denaturation firstly involves an enzymatic reduction of disulphide bonds that mainly accounts for keratin resistance (Chattaway et a/., 1963; Weary et

a/.~

1965 and Weary and Canby, 1967). However,

keratinolysis in fungi is an aerobic and oxygen dependent process and excess sulphur is excreted in an oxidized and not in a reduced form. Products of putative reduction, sulphydryl compounds like cysteine were reported by some workers (Hasija et a/., 1990; Malviya et a/., 1991, 1992a, band Rajak et a/., 1991a) but they were mostly detectable only in trace amount (Evan and Hose, 1975; Deshmukh and Agarwal, 1982, 1985 and Kunert, 1989a, b). Extracellular enzymes capable of a direct reduction of cystine combined in proteins were not found in keratinolytic fungi. ~ R-COO- + R-NH 3 +

PROTEOLYSIS: R-CO-NH-R

~ NH3 (NH3+ H 2 0 ....... NH4 + + OH-)

DEAMINATION: R-NH 3+

Cystine oxidation

sol-

<

SULPHITOLYSIS:R-SS':'R +

~ R-SS0 3- + R-S- (R-SH)

sol-

Main chemical reaction in keratinolysis by Fungi

Chester and Mathison (1963) and later on Ziegler and Bohme (1969) presented another hypothesis that the key reaction of keratinolysis in fungi 52

is deamination. It removes excess of nitrogen and allows the fungus to utilize protein as an only nutrient source. Released ammonia causes an alkalization and could denature keratin by disturbing hydrogen and ionic bonds. All keratinolytic organisms do alkalize the medium actively and this holds true also for keratinolytic fungi. Ziegler (1966) studied the physiology of decomposition of keratin over a wide range of pH and observed that optimum pH for keratinase is 9, for amylase-7.2, alkyl phosphatase-8.7, Iipase-6.8, proteinase-5.4 to 6.9. He also observed that ectoenzymes of dermatophytes are rendered inactive at pH below 4.5 and the enzymatic catabolism of keratinophilic fungi can only take place at pH above 6.9. Also Ziegler and Bohme (1969); Evan and Hose (1975); Hose and Evan (1977); Deshmukh and Agarwal (1982, 1985); Safranek and Goos (1982); Kunert (1989a, b); Hasija et a/. (1990); Malviya et at. (1991, 1992b) and Rajak et a/.

(1991a,

1992) supported this hypothesis suggesting that though

deamination and alkalization surely plays a role in keratinolysis, but they alone cannot cause significant substrate denaturation. As early as 1949 (Stahl et a/.), it was known that keratinolytic fungi are able to utilize free cystine added to a nutrient media not only as a source of sulphur but also as a source of carbon and nitrogen. Excess sulphur (26.7%) from this compound is excreted after oxidation in the form of inorganic sulphate (Ziegler and Bohme, 1977 and Malviya et a/. f 1992a). Kunert (1975) observed free sulphite in some cultures. However, in most of the experiments sulphite found in bound form "Bound-S0 3" was infact 5sulphocysteine (Cys- SS03H): Cys - SS - Cys

+ HS0 3-

~ Cys - SH

Cystine

sulphite

Cysteine

+ CyS-SS03 S-sulphocysteine

Sulphite can be liberated from S-sulphocysteine by cyanide according to the following equation: Cys - SS03 -

+ CN-

--.~

Cys - CSN

+ S03 -

This reaction was utilized for determination of both free and combined 5sulphocysteine (Kunert, 1973). Since sulphitolysis of disulphide bridges takes place in cystine combined in protein including keratin, Kunert (1995) presented a new hypothesis on bi()chemical mechanism of keratin degradation by fungi. This hypothesis postulate that sulphitolysis is a key reaction of keratinolysis. Fungi excrete sulphite and ammonia in an alkaline medium; they cleave 53

disulphide bonds of the substrate by sulphite. Thus the substrate is gradually denatured and rendered susceptible to the attack of fungal proteases (keratinases).

Evidences for sulphitolysis The studies of Kunert (1972b, c; 1973; 1975; 1976a; 1985 a, b, c; 1989a, b; 1992 and 1995) contributes that a complete hydrolysis or complete degradation of keratin can only be achieved after its denaturation by the cleavage of disulphide bonds - the main source of extra ordinary stability and resistance to proteolytic digestion. Moreover, the studies of Ruffin et al. (1976) and Weary and Canby (1967) contribute towards the evidence for sulphitolysis. The former detected

the

presence of S-

sulphocysteine in culture fluid and confirmed the role of sulphitolysis during keratin degradation by Keratinomyces ajel/oi and the latter recorded the production of 21 and 38 !-Ig/ml of cysteine by 2 strains of T. rubrum. These findings played an important part in analyzing the role of sulphitolysis during keratin degradation by keratinophilic fungi. Kunert (1981b) studied the suitability of 30 organic compounds of which 19 were sulphur-containing amino acids, as a sulphur source for the growth of dermatophyte M. gypseum. In a study of Kunert (1988b), 16 strains of dermatophytes were tested to metabolize cystine in glucose-peptone medium with a different carbon and nitrogen ratio. Cystine was utilized as a sulphur source and in addition as a source of carbon and nitrogen. In a physiologically alkaline medium the growth was fast and accompanied by an increase pH and cystine was utilized intensively. Sulphate was the main product of oxidation and sulpite was produced ata low concentration, at the beginning of growth in particular. Only traces of thio-compound (cysteine) were present in the medium. In a physiologically acid medium the growth was soon limited by a fall in pH (below 5) but cystine was continuously utilized at an identical rate. All cystine was used up by 5 species. The tendency to produce sulphite in addition

to

predominant

sulphate product.

further

increased

Concentration

of

and thiol

sulphite

was

compounds

often

the

was

also

substantially higher. Thus, dermatophytes can · utilize cystine even under conditions that do not support good growth and increase the sulphite production. In another study also, Kunert (1988a) studied the role of sulphur metabolism

in

keratin

degradation

by dermatophytes

in

a medium-

containing gelatin as the main nutritional source and supplemented with 54

cystine. All species utilized cystine not only as a sulphur source but also as a source of carbon and nitrogen and excreted excess sulphur back into the medium and the results indicated that a relationship existed between cystine metabolism and growth intensity. Rajak et al. (1991a) summarized the evidence for the presence of cystine, cysteine, inorganic thiosulphate, sulphate, keratinases and total protein as well as change in alkalinity in culture filtrate ·of 5 keratinophilic fungi. Other views supporting the role of sulphitolysis during keratin degradation includes those of Wawrzkiewicz et al. (1991) who found that dermatophytes metabolize free cystine in synthetic culture medium and utilize it not only as a source of sulphur, but also of carbon and nitrogen. According

to

Kunert

(1992)

dermatophytes

and

non-dermatophytes

metabolize free or combined cysteine as a source of sulphur and nitrogen (Ziegler et al., 1969; Kunert, 1972b, c, 1975, 1988a, band Malviya et al., 1993a, b). The products of cysteine metabolism by fungi were inorganic sulphur and other intermediate products. Kunert (1992) indicated that excess sulphur is excreted back to the medium in oxidized form as sulphate and sulphite. This sulphite reacts at neutral to alkaline pH with cystine cleaving it to cysteine and 5- sulphocysteine: Cys -S - S - Cys

+ HS0 3-

• Cys-SH

+ CyS-SS03

Malviya et al. (1992c, d) in fact, found that keratinase enzyme was not detected in the culture medium until more than half of supplied keratin had been degraded. This indicated a non-enzymatic degradation of keratin but the presence of soluble products of sulphitolysis such as peptides and amino acids in the culture fluid (Weary and Canby, 1967; Kunert, 1972b, c, 1973, 1976a; Ruffin et al., 1976; Rajak et al., 1991a, b, 1992 and Malviya et al., 1993a, b, 1994) is a further indication of disulphide breakdown. Deshmukh and Agarwal (1982, 1985) and Lin et al. (1992) also estimated the presence of protein, peptide, amino acids but they were mostly detected in trace concentrations. The reason for detecting intermediate compounds in low concentration is due to the preferential utilization of cysteine as a source of sulphur by fungi (Kunert, 1987). Malviya et al. (1993a, b) also suggested that the amount of residual cysteine continuously declined in the culture fluid, obviously due to its utilization in fungal metabolism. However, microbial capacity for sulphitolysis differs among different microorganisms due to the differential parasitic potential. 55

Keratinophilic' fungi as a tool for biotechnological applications A number of microorganisms produce enzymes, which are involved primarily in the degradation of macromolecules to unit capable of being taken into the living cells. These proteases are capable of cleaving proteins into smaller units such as peptides and amino acids, which are further used as a reagent

in

laboratory, food

industry, ' leather industry, feather

degradation and also in pharmaceutical industry. The production of different enzymes by keratinophilic fungi is of immense value for their successful survival and subsequent hydrolysis of keratin. They have extracellular enzymes which are useful in eco-ethical technology such as for digestive processes (amylase), degradation of cellulolytic waste from pulp and paper industry ' (cellulase), fermentation and enhancement of dairy products (lipase) for de-hairing of skin and feather degradation (keratinase) , hence forth, removes the soil contamination (Kavitha et al., 1997). Feather keratin is a waste from poultry industry. The feathers, wh ich are hydrolyzed by mechanical or chemical treatments can be converted to feedstuffs, fertilizers, glues and foils or used for the production of amino acids and peptides. Because of environmental consideration the use of keratinolytic enzymes in the production of amino acids and peptides is becoming attractive for biotechnological applications. With the help of these enzymes feathers could be converted to defined products such as rare amino acids, serine, cysteine and proline. This enzymatic process is advantageous over commercial method, as large amount of salts, which have to be separated from the end product, would not be produced . The production of keratinases has been a domain of mesophilic fungi and actinomycetes until now (Yu et al., 1969a, b, 1972; Kunert, 1972b, c, 1973; Kunert and Krajci, 1981; Wawrzkiewicz et al., 1987; Sinha et al., 1991 and Bockle et al., 1995). The amino acids released by keratinophilic fungi finds application in various ways such as flavouring agents, for improvement of taste of food , as therapeutic agents, as an anti-oxidant, as a dough conditioner (cysteine and cystine) and as a food supplement (methionine). Chattejee and Chaterjee (1997) made an attempt to study the microbial production of L-Iysine. Parihar and Kushwaha (1999) used higher concentration of hen's feather during

keratin

degradation

and

proved

that the

process

of keratin

decompostion plays a very important role in energy transformation and nutrient cycling in soil. Parihar and Kushwaha (2000) also conducted a 56

survey of keratinophilic fungi and used them as a tool for hen's feather utilization. William and Shih (1989) added some methane producing, cellulolytic, feather degrading, aerobic microbial population in the poultry waste

digestor . and

observed

enhancement

in

feather

degradation.

Deshmukh and Agarwal (1982) studied enzyme-producing capabilities of keratinophilic fungi. He carried out a study to observe amylase, protease, lipase, urease, chitinase and phosphatase. Singh and Agarwal (1987) studied protease production by certain keratinophilic fungi. The hydrolyzing action of protease plays an important role in the preparation of various enzymatic products, which have a wide application in the field of brewery, textiles, dairy,

pharmaceuticals and

leather industry. The predominance of a

particular type of proteinase may depend upon the nature of the growth medium that is employed. Kellehar et al. (2002) showed that the waste from the poultry industry has a high nutritional value. Microbial degradation helps convert .this environmental burden into various by-products used as an organic fertilizer, thus recycling nutrients such as nitrogen, phosphorus and potassium in nature. All these evidences justify the biotechnological importance of these fungi for improving the utilization of poultry waste feathers via microbial technology.

57