During a survey of the occurrence of Malassezia species in the external ear canals of dogs with chronic otitis externa, lipid-dependent Malassezia species were isolated in three dogs. These species were identified as Malassezia furfur and M. obtusa but showed atypical assimilation patterns. To our knowledge, this is the first report of the isolation of lipid-dependent species of the genus Malassezia in association with canine otitis

Abarca, M. L.

Cabañes, F. J.

Crespo, M. J.

PubMed

Crespo Domínguez, Ma. Jesús

Abarca Salat, Maria Lourdes

Cabañes Sáenz, Francisco Javier

Diposit Digital de Documents de la UAB

JOURNAL OF CLINICAL MICROBIOLOGY,0095-1137/00/$04.0010June 2000, p. 2383–2385 Vol. 38, No. 6Copyright © 2000, American Society for Microbiology. All Rights Reserved.Atypical Lipid-Dependent Malassezia Species Isolatedfrom Dogs with Otitis ExternaM. J. CRESPO, M. L. ABARCA, AND F. J. CABAN˜ES*Departament de Patologia i Produccio´ Animals (Microbiologia), Facultat de Veterina`ria,Universitat Auto`noma de Barcelona, 08193 Bellaterra (Barcelona), SpainReceived 28 December 1999/Returned for modification 26 February 2000/Accepted 25 March 2000During a survey of the occurrence of Malassezia species in the external ear canals of dogs with chronic otitisexterna, lipid-dependent Malassezia species were isolated in three dogs. These species were identified asMalassezia furfur and M. obtusa but showed atypical assimilation patterns. To our knowledge, this is the firstreport of the isolation of lipid-dependent species of the genus Malassezia in association with canine otitis.Species of the genus Malassezia inhabit the skin of humansand many other warm-blooded vertebrates (20). Their mostimportant physiological feature is their lipophilic nature, be-cause they have the property of using lipids as a source ofcarbon. In fact, with the exception of Malassezia pachydermatis,the remaining species of the genus have an absolute require-ment in vitro for supplementation with long-chain fatty acids inthe culture medium.The taxonomy of these lipophilic fungi has always been amatter of controversy. A few years ago, M. furfur (Robin)Baillon 1889, M. pachydermatis (Weidman) Dodge 1935, andM. sympodialis Simmons and Gue´ho 1990 were the only rec-ognized species. Recently, the genus has been revised on thebasis of morphology, ultrastructure, physiology, and molecularbiology and enlarged to seven species that include the threetaxa mentioned above and four new taxa: M. globosa, M. ob-tusa, M. restricta, and M. slooffiae (12).These yeasts are etiological agents of chronic and superficialskin disorders. In addition, their importance as emergentpathogens in humans is increasing because they have beenidentified as causative agents of sepsis in immunocompromisedpatients (1) and neonates receiving parenteral lipid alimenta-tion (23). All lipid-dependent species can be isolated fromnormal and diseased human skin, and they can become patho-genic under the influence of predisposing factors. They areassociated with several diseases, such as pityriasis versicolor,folliculitis, seborrheic dermatitis, and some forms of atopicdermatitis and even systemic infection (11). M. pachydermatiscan be isolated from the skin and mucosae of a variety of birdsand mammals, including dogs and cats (9, 14). Although thisspecies is mainly adapted to nonhuman animals, it has beenreported to cause nosocomial systemic infection in humans(19, 24). This yeast can be transmitted by human health-careworkers from their pet dogs to their neonatal patients (5). It isthe most common yeast that contributes to chronic otitis ex-terna in dogs (22).During a survey of the occurrence of Malassezia species inthe external ear canals of dogs with chronic otitis externa,lipid-dependent Malassezia species were isolated in three dogs.To our knowledge, this is the first report of the isolation oflipid-dependent species of the genus Malassezia in associationwith canine otitis.Microbiology. Swabs from the external ear canals of 57 petdogs (29 male and 28 female) with chronic otitis externa wereobtained for microbiologic examination. Each sample was in-oculated onto the following media: blood agar, MacConkeyagar, Sabouraud glucose agar (SGA), SGA supplemented witholive oil (10 ml/liter), and Leeming’s medium (10 g of peptone,5 g of glucose, 0.1 g of yeast extract, 4 g of desiccated ox bile,1 ml of glycerol, 0.5 g of glycerol monostearate, 0.5 ml ofTween 60, 10 ml of whole cow’s milk, 12 g of agar per liter [pH6.2]) (17). The last three media were made for mycologicalexamination and contained 0.05% chloramphenicol and 0.05%cycloheximide. For cytological examination, specimens wereheat fixed and stained with Gram and Diff-Quick stains. Platesof SGA, SGA supplemented with olive oil, and Leeming’smedium were incubated at 35°C and examined after 3, 5, 7, and14 days. Plates of blood agar and MacConkey agar were incu-bated at 37°C with 5% CO2 for 3 days. When growth on SGAsupplemented with olive oil and/or Leeming’s medium wasdetected, five different colonies were selected from each me-dium and subcultured on SGA to determine their lipid depen-dence. M. pachydermatis was identified by morphology micro-scopically and by the ability to grow on SGA. The identificationof the lipid-dependent yeasts was based on the ability to usecertain polyoxyethylene sorbitan esters (Tweens 20, 40, 60, and80), as described by Gue´ho et al. (12), and the Tween diffusiontest proposed by Guillot et al. (15). The Cremophor EL as-similation test and the splitting of esculin described by Mayseret al. (18) were used as additional tests for the differentiationof the species M. furfur, M. slooffiae, and M. sympodialis.Gram and Diff-Quick stains revealed the presence ofMalassezia cells in 39 dogs (68.4%). Bacteria and Malasseziawere seen in 30 of these dogs (76.9%), and Malassezia alonewas detected in 9 dogs (23.1%). More than 10 yeasts perhigh-power field were observed in 14 of the 39 dogs (24.6%),and less than 10 yeasts per high-power field were detected in 25of them (43.8%), but 5 of these last samples were negative forMalassezia in cultures. Bacteria alone were seen in 16 dogs(28.1%), and for 2 dogs (3.5%), the cytological examinationwas negative. Microbiological cultures were positive for 54dogs (94.7%). Pure cultures for bacteria were obtained for 15dogs (26.3%). Malassezia species were isolated from 39 dogs(68.4%), but for 5 of these dogs, typical Malassezia cells werenot observed in cytological examinations. Yeasts belonging toother genera were not isolated. M. pachydermatis was the onlyyeast isolated from 36 dogs (63.1%). In 27 of these dogs (75%),* Corresponding author. Mailing address: Departament de Patolo-gia i Produccio´ Animals (Microbiologia), Facultat de Veterina`ria, Edi-fici V, Room V0-285, Universitat Auto`noma de Barcelona, 08193-Bellaterra (Barcelona), Spain. Phone: 34 93 581 17 49. Fax: 34 93 58120 06. E-mail: javier.cabanes@uab.es.2383 on June 8, 2018 by UAB/FAC VETERINARAhttp://jcm.asm.org/Downloaded from the yeast was mixed with bacteria, and only in 9 dogs (25%)was the yeast obtained in pure cultures. Lipid-dependent spe-cies were obtained in mixed cultures with bacteria from threedogs (5.3%). In one of them, the species M. pachydermatis wasalso isolated.The lipid-dependent isolates obtained from two dogs formedcream, mat, and smooth colonies with a convex elevation.Their textures were soft, and their average diameters were 3.6to 5.4 mm on modified Dixon’s agar (36 g of malt extract, 6 gof peptone, 20 g of desiccated ox bile, 10 ml of Tween 40, 2 mlof glycerol, 2 ml of oleic acid, 12 g of agar per liter [pH 6.0])(12) after 7 days of incubation at 32°C. The cells were cylin-drical and oval (1.7 to 2.1 mm by 2.9 to 4.3 mm). Buds wereformed on a broad base, and short filaments were not observedin any sample. The catalase reaction was positive. The yeastsutilized the four Tweens, grew on glucose-peptone agar with0.5% Tweens 40 and 60, 0.1% Tween 80, and 10% Tween 20,and were able to split esculin in 2 to 3 days, as was the typestrain M. furfur CBS 1878 under the same conditions. Theseisolates did not grow around the Cremophor EL-containingwell at 7 days of incubation. However, an inhibition area mea-suring 20 to 25 mm in diameter around the well was sur-rounded by a ring of tiny colonies after 10 days of incubation(Fig. 1). This pattern clearly differed from the complete diskformed by colonies of the M. furfur type strain at 7 days (Fig.2). To confirm that these yeasts really assimilated CremophorEL, this test was performed again on a single plate, and thesame results were obtained at 10 days of incubation. To thisend, we confirmed that these lipid-dependent species assimi-lated Cremophor EL, but not at the high concentration of thiscompound present near the well. According to these findings,the yeasts had characteristics identical to those of M. furfur,with the exception of the growth pattern observed for theCremophor EL test.The lipid-dependent yeasts isolated from the third dogformed cream, smooth, and flat colonies with average diame-ters of 1.0 to 1.4 mm on modified Dixon’s agar after 7 days ofincubation at 32°C. Their textures were sticky, buds wereformed on a broad base, and the cells were cylindrical (1.7 to2.1 mm by 2.4 to 2.8 mm). The catalase reaction was positive,the isolates did not utilize either the four Tweens or Cremo-phor EL, and the splitting of esculin was positive in 2 to 3 days,as it was for the type strain M. obtusa CBS 7876 under the sameconditions. Nevertheless, they were able to grow on glucose-peptone agar with 0.5% Tweens 40 and 60, a result whichdiffered from the identification of this species proposed byGue´ho et al. (12). According to these findings, these lipid-dependent species had the same characteristics as the speciesM. obtusa, except for their growth on glucose-peptone agarwith 0.5% Tweens 40 and 60.Discussion. The importance of M. pachydermatis in dogs hasbeen extensively reported (13, 16). This species can play animportant role in chronic dermatitis and otitis externa in car-nivores, especially in dogs. This yeast has an opportunisticnature, and it may become pathogenic with any alteration inthe skin surface microclimate or in host defense. Canine otitisexterna and seborrheic dermatitis are frequently associatedwith large numbers of M. pachydermatis (22). The frequency ofisolation of this species from the external ear canal is reportedto be 15 to 49% for healthy dogs and may increase up to 50 to83% for dogs with otitis externa (10).Classically, lipid-dependent species were related to humanskin only, but it is now known that the skin of different animalsalso can be colonized by lipid-dependent species in addition toM. pachydermatis (14). M. slooffiae seems to represent a largeproportion of the cutaneous yeast flora in pigs, and it has beenisolated from normal skin in sheep and goats as well (12). Thefollowing lipid-dependent species have been isolated fromhealthy bovines (8, 12): M. globosa, M. furfur, M. slooffiae, M.obtusa, and M. sympodialis. The presence of lipid-dependentspecies in carnivores has been recently demonstrated. M. sym-podialis (3), M. globosa (4), and M. furfur (6) have been de-scribed to colonize the skin and mucosae of healthy cats. M.furfur and M. sympodialis also have been reported from caninespecimens tested by identification techniques such as the as-similation of Cremophor EL, the splitting of esculin, and pig-ment synthesis (21).The role of lipid-dependent species in human skin is wellFIG. 2. Cremophor EL assimilation test of the type strain M. furfur CBS1878. Note that a complete disk formed by colonies is observed around the wellat 7 days of incubation.FIG. 1. Cremophor EL assimilation test of an atypical M. furfur isolate. Notethat an inhibition area around the well is surrounded by a ring of tiny colonies at10 days of incubation.2384 NOTES J. CLIN. MICROBIOL. on June 8, 2018 by UAB/FAC VETERINARAhttp://jcm.asm.org/Downloaded from documented. They are commensal yeasts that may becomeetiological agents of cutaneous and systemic diseases. How-ever, very little is known about their role in nonhuman animalskin. A skin disorder in goats associated with Malassezia cellsmorphologically different from M. pachydermatis cells was de-scribed on the basis of histological techniques, but the isolationin vitro of the casual agent was unsuccessful (2). More recently,lipid-dependent species have been isolated in bovines withotitis externa (M. globosa, M. slooffiae, M. furfur, and M. sym-podialis) (8), and two cases of feline otitis externa associatedwith M. sympodialis have been reported from our laboratory(7).In this study, three atypical lipid-dependent yeasts have beenisolated. To our knowledge, this is the first isolation of lipid-dependent species in association with canine otitis externa.Lipid-dependent yeasts probably have a pathogenic mecha-nism similar to that of M. pachydermatis in the skin. They aremembers of the cutaneous flora of some animals, and theymight become pathogenic under the same predisposing factors.However, more studies would be required to determine theirrole in animal skin. For this reason, culture media with lipidsources, such as SGA supplemented with olive oil, Leeming’smedium, or modified Dixon’s agar, should be used in additionto media without lipid sources. Our finding of lipid-dependentisolates indicates that canine otitis externa can be associatedwith lipid-dependent Malassezia species in addition to the non-lipid-dependent species M. pachydermatis.On the other hand, the identification system for the lipid-dependent species based on the ability to use certain polyoxy-ethylene sorbitan esters (Tweens 20, 40, 60, and 80), the Cre-mophor EL assimilation test, and the splitting of esculin wasnot able to identify our canine lipid-dependent isolates. Somedifferences in the assimilation patterns were observed on com-parison with the type strain patterns. It is likely that the num-ber of lipid-dependent species that do not fit the described typespecies profiles increases as long as further isolates are ob-tained from different hosts. In these such situations, comple-mentary tests, including molecular techniques, would be re-quired to differentiate atypical lipid-dependent isolates. On theother hand, the possibility of some variations in assimilationpatterns within a species should be considered.We thank Autonomous University of Barcelona Veterinary Teach-ing Hospital and various veterinary clinics from the Barcelona regionfor the samples kindly provided for this investigation.REFERENCES1. Barber, G. R., A. E. Brown, T. E. Kiehn, F. F. Edwards, and D. Armstrong.1993. Catheter-related Malassezia furfur fungemia in immunocompromisedpatients. Am. J. Med. 95:365–370.2. Bliss, E. L. 1984. Tinea versicolor dermatomycosis in the goat. J. Am. Vet.Med. Assoc. 184:1512–1513.3. Bond, R., R. M. Anthony, M. Dodd, and D. H. Lloyd. 1996. Isolation ofMalassezia sympodialis from feline skin. J. Med. Vet. Mycol. 34:145–147.4. Bond, R., S. A. Howell, P. J. Haywood, and D. H. Lloyd. 1997. 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Atypical Lipid-Dependent Malassezia Species Isolated from Dogs with Otitis Externa

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Atypical Lipid-Dependent Malassezia Species Isolated from Dogs with Otitis Externa

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