Mycopathologia 149: 131–135, 2000. © 2001 Kluwer Academic Publishers. Printed in the Netherlands.
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Extracellular enzymatic activity of Malassezia spp. isolates Francesca Mancianti, Antonello Rum, Simona Nardoni & Michele Corazza Dipartimento di Patologia Animale, Profilassi ed Igiene degli Alimenti, Universit´a di Pisa, Viale delle Piagge, 2, I 56100 Pisa, Italy Received 6 September 2000; accepted 26 November 2000
Abstract Extracellular enzymatic activity of different species of Malassezia spp was evaluated. Thirty-three isolates of animal origin (dogs and cats) and stock culture samples were studied. Twenty isolates of M. pachydermatis, 8 of M. furfur, 2 of M. sympodialis and M. globosa and one of M. restricta, M. obtusa and M. slooffiae were examined. The enzymatic activity was investigated using Api Zym system. The enzymatic patterns showed light differences. Esterase lipase, Phosphatase acid and Naphtol-AS-BI-phosphohydrolase were produced in significant amounts from most isolates excepted for M. restricta, confirming the limited enzymatic activity of this species. Data obtained from the other new species described after the revision of the genus, appear to be quite homogeneous. Dixon’s broth appeared to be a valid medium for the growth of all Malassezia spp. Key words: Malassezia spp., enzymatic activity, Api Zym.
Introduction The genus Malassezia is composed of lipophilic basidiomycetous yeasts which has been recently reviewed and appears to consist of seven species [1, 2]. The species Malassezia furfur, Malassezia sympodialis, Malassezia globosa, Malassezia obtusa, Malassezia slooffiae and Malassezia restricta are lipid-dependent, while Malassezia pachydermatis is the sole non strictly lipid-dependent, not inhibited by 0.05% of cicloheximide. For this reason M. pachydermatis has been often isolated from healthy or affected skin of animals [3, 4, 5]. In the last years lipophilic species also, such as M. globosa and M. furfur, have been isolated from cats [6], M. sympodialis from canine and feline specimens [7] and M. globosa, M. slooffiae, M. furfur and M. sympodialis from bovines affected by otitis, while M. obtusa and M. pachydermatis were isolated only from healthy bovines [8]. M. globosa, M. sympodialis and M. restricta were isolated also from human normal skin showing different body site locations [9]. M pachydermatis was associated with systemic infections of humans [10], isolated from an uditive smear [11], described as cause of infection for infants in neonatal intensive care units [12, 13] and
isolated from 12% on human healthy skin [14] on the head and palm. In our experience M. furfur, M. pachydermatis and M. sympodialis are the species more frequently isolated from dogs with otitis and allergic dermatitis and their identification based on biochemical differences is suitable also in routine diagnosis [15, 16, 17]. Only little is known about the biochemical patterns of these yeasts and in vitro reports regard the lipolytic activities of M. furfur and M. pachydermatis.. In this paper we report the cellular enzymatic activity of different species of Malassezia isolated from affected animals (dogs and cats) and coming from stock collection in order to evaluate an interspecific and/or intraspecific variability. Materials and methods Isolates of Malassezia Thirty-three isolates of Malassezia were used. The yeasts consisted of 20 specimens of M. pachydermatis, 8 of M. furfur, 2 of M. sympodialis and M. globosa, and one of M. restricta, M. obtusa and M. slooffiae.
132 12 dogs and 2 cats. More precisely the isolates of M. pachydermatis and M. sympodialis were obtained from canine cutaneous and/or otologic specimens. The specimens identified as M. furfur were isolated from three dogs and one cat. One of the samples of M. globosa was of feline origin, coming from an otologic specimen. The species were isolated onto Dixon medium [2] and identified on the basis of their growth on dermatophyte selective medium (M. pachydermatis), the production of dark colonies on Dixon’s modified medium (M. furfur) [7] and their capability of splitting esculine (M. sympodialis) [16]. M. globosa was recognised for its incapability to grow onto 0.5% tween 40 and at 40 ◦ C with respect to M. slooffiae, for the presence of catalase with respect to M. restricta and on the basis of its microscopical appearance with respect to M. obtusa. Other specimens were from culture stocks from the Institut Pasteur, France. A week later the yeasts were inoculated into a Dixon broth prepared as the solid medium, without agar. An inoculum of about 105 cells/ml−1 was added to about 10 ml of medium and the tubes were incubated at 32 ◦ C for 15 days. When the yeasts had shown a good growth on the surface of the medium and on the bottom of the culture tube, the liquid phase was employed for enzymatic studies. Determination of enzymatic activities The inocula were processed as previously described for Microsporum canis culture extracts [18]. Briefly, 65 µl of the liquid phases from incubated cultures were filtered through 0.45 µm (Nalgene, USA) and inoculated in each cupule of Api Zym strips (BioMeneux, Roma, Italy) following the manufacturer’s instructions. The following enzymatic activities were tested: alkaline phosphatase, esterase (C4), esterase lipase (C8), lipase (C14), leucine arylamidase, valine arylamidase, cystine arylamidase, trypsin, chimotrypsin, acid phosphatase, naphtol-AS-BIphosphohydrolase, α-galactosidase, β-galactosidase, β-glucuronidase, α-glucosidase, β-glucosidase, N-acetyl-β-glucosaminidase, α-mannosidase and α-fucosidase. The strips were incubated at 37 ◦ C for 4 hours and colour reagents Zym A (Tris-hydroxymethylaminomethane, 37% Hydrochloric acid and Lauryl sulphate in distilled water) and Zym B (Fast Blue BB in 2 methoxyethanol) were added to each cupule. The approximate number of free nanomoles was appreciated from
the color strength and indicated with an intensity ranging from 0 to 5, with 0 corresponding to 0 nanomolcs of hydrolysed substrate, 1 to 5 nanomoles, 2 to 10 nanomoles, 3 to 20 nanomoles, 4 to 30 nanomoles i to ≥ 40 nanomoles. All the tests were performed in duplicate and negative controls were performed by using uninoculated medium.
Results The enzymatic patterns showed light differences. M. pachydermatis showed 15 enzymalic activities, with intensities ranging from 1 to 5. Eleven isolates were positive for 5 enzymes, 3 for 4, 2 for 8 and 1 respectively for 3, 6, 7 and 9. Leucine arylamidase, trypsin, chymotrypsin, α-galactosidase, α-glucosidase and α-fucosidase never occurred. M. furfur secreted 8 enzymatic activities, their intensities also ranged from 1 to 5. Three isolates secreted 6 enzymes, 3 produced 5 enzymes and I showed 2 and 1 enzymatic activities respectively. Leucine arylamidase, valine arylamidase, cystine arylamidase, trypsin, chymotrypsin, α-galactosidase, β-galactosidase, α-glucosidase, β-glucosidase, βmannosidase and α-fucosidase were never secreted. The two strains of M. sympodialis produced 5 enzymes. The stock collection sample of M. globosa produced 3 enzymes, while the feline isolate showed 4 enzymatic activities. M. restricta secreted naphtolAS-BI-phosphohydrolase only, M. obtusa secreted 4 enzymes and M. slooffiae showed a strong enzymatic activity and secreted 5 enzymes. The higher intensities were related to esterase lipase (C8), phosphatase acid and naphtol-AS-BI-phosphohydrolase for M. pachydermatis, M. furfur, M. sympodialis and for the isolate of M. slooffiae. The ability of the examined samples to produce extracellular enzymes is reported in Tables 1, 2 and 3.
Discussion Our data confirm that Malassezia spp. as a pathogen yeast is more frequently reported in dogs than in cats [15]. In both species these fungi are part of the normal flora, however they have been isolated also in animals showing otitis [4] and dermatitis [20].
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Table 1. Enzymatic activity of M. pachydermatis
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
2 2 1 1 1 1 1 1 1 1 1
2
1 1 1 1 1 1 1
2 1
4 4 1 5 1 4 1 2 2 3 4 1 1 2 2 4 2 4 4 2
2 1
3 3
3 3 1 4 3 3 2 1 2 3 1 1 2 2 4 3 3 3 4 4
2 1 1 1 1
1 1 1 1 1 2 1 1
1 1
2 2 1 2 1 2 2 2 2 2 2 2 2 4 2 2 2 2
1
1
1
1
1 2
1 2
1 2
Table 2. Enzymatic activity of M. furfur Phosphatase alkalyne 1 2 3 4 5 6 7 8
Esterase
1 1
Esterase Lipase
Lipase
Phosphatase acid
Naphtol-AS-BIphosphohydrolase
βglucuronidase
1 1 1
4 3 2 1
1 1 1
1 1 5
1 1 5
2 1 4
2 3 2 2 2 2 4
1 2 2 2 2 2 4
N-acetyl-β glucosaminidase
1 1
1
134 Table 3. Enzymatic activity of other Malassezia species Phosphatase alkalyne
Esterase
Malassezia sympodialis 1 1 2 2 2 Malassezia globosa 1 1 2 2 1 Malassezia restricta 1 1 Malassezia obtusa 1 1 1 Malassezia slooffiae 1
Esterase Lipase
Phosphatase acid
Naphtol-AS-BIphosphohydrolase
1 3
4 1
4 1
1 3
3
2 3
βglucuronidase
βglucosidase
3
3
1 3
4
2
4
4
2
All Malassezia strains tested in the present work grew on Dixon’s broth. To obtain an available enzymatic production from the isolates, a liquid medium was requested and Dixon’s broth was able to support an abundant growth of the isolates. This culture method could be used for routine purposes when a great number of cells are needed. To the best of our knowledge, this is the first report of the use of Dixon’s broth for the culture of Malassezia. In our work oleic acid was used as lipidic supplement: it is important to remark that M. pachydermatis, which is the only species not recognized as strictly lipid-dependent, showed a better growth when oleic acid was present in the medium. According to standard descriptions this species, most frequently recovered from domestic carnivora, grows readily on lipid-unsupplemented complex media but may require fatty acids for growth on simple defined media [10]. M. furfur and M. pachydermatis are known to produce several different enzymes [21, 22]. A strong extracellular hydrolase activity has been described from human clinical specimens of M. furfur [21]. In accordance with these results, in the present study naphtol-AS-BI-phosphohydrolase was secreted from all the isolates but one. Six on eight isolates of M. furfur produced different amounts of lipase: this result agrees with Muhsin et al. [22], reporting an high lipase and phospholipase production for this yeast. The role of lipase and lipoxygenase due to M. furfur was demonstrated both in vivo and in vitro [23, 24]. Phospholipase appears to be produced from pathogenic strains [25] and it is able to hydrolyze lecithin producing free fatty acids, essential for fungus growth [26]. Both lipase and phospholipase are suggested to
maintain the function of the fungal cell membrane and to favourize the invasion of host tissue [27]. Ran et al. [24] studied biochemical characteristics and properties of lipase in M. furfur, concluding that this enzyme, which is recognized as associated with the surface of yeast cell [23], has an important role with regard to pathogenicity. Esterase activity with lauric, palmitic and stearic acid was revealed in all M. pachydermatis from canine otitis, while oleic acid esters were hydrolized by only 42 of the 80 examined strains [28]. Excepting M. restricta, no strictly interspecific variabilities were observed. Among the different isolates belonging to the same species, the variability was very weak. All the yeasts but M. restricta produced different amounts of esterase lipase (C8). M. restricta is the only lipid-dependent taxon in which catalase reaction does not appear: the specific epithet restricta refers to its limited growth in vitro. Our results reflect the limited enzymatic activity of this species [2]. Beside the reduced number of isolates belonging to M. sympodialis, M. slooffiae, M. globosa and M. obtusa the results appears to be quite homogeneous. The present finding cannot be supported with results obtained by other authors, because only little data regarding these new species are available in literature. Several studies about lipases of Malassezia have been done before the revision of the genus, when these yeasts were believed to be only 2 species. Probably these results should be evaluated with best attention, because they could have been obtained by mixed cultures. A work performed by Raabe et al. [7] reported that Malassezia yeasts in mixed cultures are frequently found both in animals and in humans. On 47 veterinary specimens (dogs and cats) 9 were recognised
135 as pure cultures (3 of M. pachydermatis and 6 of M. sympodialis respectively); in 14 cases M. furfur and M. sympodialis were demonstrated together with M. pachydermatis. Although a comparison of pathogenicity with both patterns and levels of esoenzyme production is not present in this work, it is believed that these new observations on the extracellular enzymatic activity of different species of the yeast could provide data for further investigations on the pathogenic role of enzymes secreted by Malassezia spp.
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Address for correspondence: Dr Francesca Mancianti, Dipartimento di Patologia Animale, Viale delle Piagge, 2 Phone: (39) 50 544260; Fax: (39) 50 5406044; E-mail:
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