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Journal of Clinical Microbiology, June 2000, p. 2383-2385, Vol. 38, No. 6
0095-1137/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Atypical Lipid-Dependent Malassezia
Species Isolated from Dogs with Otitis Externa
M. J.
Crespo,
M. L.
Abarca, and
F. J.
Cabañes*
Departament de Patologia i Producció
Animals (Microbiologia), Facultat de Veterinària, Universitat
Autònoma de Barcelona, 08193 Bellaterra (Barcelona), Spain
Received 28 December 1999/Returned for modification 26 February
2000/Accepted 25 March 2000
 |
ABSTRACT |
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.
| |
TEXT |
Species of the genus
Malassezia inhabit the skin of humans and many other
warm-blooded vertebrates (20). Their most important physiological feature is their lipophilic nature, because they have the
property of using lipids as a source of carbon. In fact, with the
exception of Malassezia pachydermatis, the remaining species
of the genus have an absolute requirement in vitro for supplementation
with long-chain fatty acids in the culture medium.
The taxonomy of these lipophilic fungi has always been a matter of
controversy. A few years ago, M. furfur (Robin) Baillon 1889, M. pachydermatis (Weidman) Dodge 1935, and M. sympodialis Simmons and Guého 1990 were the only recognized
species. Recently, the genus has been revised on the basis of
morphology, ultrastructure, physiology, and molecular biology and
enlarged to seven species that include the three taxa mentioned above
and four new taxa: M. globosa, M. obtusa,
M. restricta, and M. slooffiae (12).
These yeasts are etiological agents of chronic and superficial skin
disorders. In addition, their importance as emergent pathogens in
humans is increasing because they have been identified as causative agents of sepsis in immunocompromised patients (1) and
neonates receiving parenteral lipid alimentation (23). All
lipid-dependent species can be isolated from normal and diseased human
skin, and they can become pathogenic under the influence of
predisposing factors. They are associated with several diseases, such
as pityriasis versicolor, folliculitis, seborrheic dermatitis, and some
forms of atopic dermatitis and even systemic infection (11).
M. pachydermatis can be isolated from the skin and mucosae
of a variety of birds and mammals, including dogs and cats (9,
14). Although this species is mainly adapted to nonhuman animals,
it has been reported to cause nosocomial systemic infection in humans
(19, 24). This yeast can be transmitted by human health-care
workers from their pet dogs to their neonatal patients (5).
It is the most common yeast that contributes to chronic otitis externa
in dogs (22).
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. To our knowledge, this is the first report of the isolation of
lipid-dependent species of the genus Malassezia in
association with canine otitis.
Microbiology.
Swabs from the external ear canals of 57 pet
dogs (29 male and 28 female) with chronic otitis externa were obtained
for microbiologic examination. Each sample was inoculated onto the
following media: blood agar, MacConkey agar, Sabouraud glucose agar
(SGA), SGA supplemented with olive 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 of Tween 60, 10 ml of whole cow's
milk, 12 g of agar per liter [pH 6.2]) (17). The last
three media were made for mycological examination and contained 0.05%
chloramphenicol and 0.05% cycloheximide. For cytological examination,
specimens were heat fixed and stained with Gram and Diff-Quick stains.
Plates of SGA, SGA supplemented with olive oil, and Leeming's medium
were incubated at 35°C and examined after 3, 5, 7, and 14 days.
Plates of blood agar and MacConkey agar were incubated at 37°C with
5% CO2 for 3 days. When growth on SGA supplemented with
olive oil and/or Leeming's medium was detected, five different
colonies were selected from each medium and subcultured on SGA to
determine their lipid dependence. M. pachydermatis was
identified by morphology microscopically and by the ability to grow on
SGA. The identification of the lipid-dependent yeasts was based on the
ability to use certain polyoxyethylene sorbitan esters (Tweens 20, 40, 60, and 80), as described by Guého et al. (12), and
the Tween diffusion test proposed by Guillot et al. (15).
The Cremophor EL assimilation test and the splitting of esculin
described by Mayser et al. (18) were used as additional tests for the differentiation of the species M. furfur,
M. slooffiae, and M. sympodialis.
Gram and Diff-Quick stains revealed the presence of
Malassezia cells in 39 dogs (68.4%). Bacteria and
Malassezia were seen in 30 of these dogs (76.9%), and
Malassezia alone was detected in 9 dogs (23.1%). More than
10 yeasts per high-power field were observed in 14 of the 39 dogs
(24.6%), and less than 10 yeasts per high-power field were detected in
25 of them (43.8%), but 5 of these last samples were negative for
Malassezia in cultures. Bacteria alone were seen in 16 dogs
(28.1%), and for 2 dogs (3.5%), the cytological examination was
negative. Microbiological cultures were positive for 54 dogs (94.7%).
Pure cultures for bacteria were obtained for 15 dogs (26.3%).
Malassezia species were isolated from 39 dogs (68.4%), but
for 5 of these dogs, typical Malassezia cells were not
observed in cytological examinations. Yeasts belonging to other genera
were not isolated. M. pachydermatis was the only yeast
isolated from 36 dogs (63.1%). In 27 of these dogs (75%), the yeast
was mixed with bacteria, and only in 9 dogs (25%) was the yeast
obtained in pure cultures. Lipid-dependent species were obtained in
mixed cultures with bacteria from three dogs (5.3%). In one of them,
the species M. pachydermatis was also isolated.
The lipid-dependent isolates obtained from two dogs formed cream, mat,
and smooth colonies with a convex elevation. Their
textures were soft,
and their average diameters were 3.6 to 5.4
mm on modified Dixon's
agar (36 g of malt extract, 6 g of peptone,
20 g of
desiccated ox bile, 10 ml of Tween 40, 2 ml of 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 cylindrical and oval
(1.7 to 2.1 µm by 2.9 to 4.3 µm). Buds were formed on a broad
base, and
short filaments were not observed in any sample. The
catalase reaction
was positive. The yeasts utilized the four Tweens,
grew on
glucose-peptone agar with 0.5% 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 type strain
M. furfur CBS 1878 under the
same
conditions. These isolates did not grow around the Cremophor
EL-containing well at 7 days of incubation. However, an inhibition
area
measuring 20 to 25 mm in diameter around the well was surrounded
by a
ring of tiny colonies after 10 days of incubation (Fig.
1).
This pattern clearly differed from
the complete disk formed by
colonies of the
M. furfur type
strain at 7 days (Fig.
2). To confirm
that these yeasts really assimilated Cremophor EL, this test was
performed again on a single plate, and the same results were obtained
at 10 days of incubation. To this end, we confirmed that these
lipid-dependent species assimilated Cremophor EL, but not at the
high
concentration of this compound 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 the Cremophor EL test.
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FIG. 1.
Cremophor EL assimilation test of an atypical M. furfur isolate. Note that an inhibition area around the well is
surrounded by a ring of tiny colonies at 10 days of incubation.
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FIG. 2.
Cremophor EL assimilation test of the type strain
M. furfur CBS 1878. Note that a complete disk formed by
colonies is observed around the well at 7 days of incubation.
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|
The lipid-dependent yeasts isolated from the third dog formed cream,
smooth, and flat colonies with average diameters of 1.0
to 1.4 mm on
modified Dixon's agar after 7 days of incubation
at 32°C. Their
textures were sticky, buds were formed on a broad
base, and the cells
were cylindrical (1.7 to 2.1 µm by 2.4 to
2.8 µm). The catalase
reaction was positive, the isolates did
not utilize either the four
Tweens or Cremophor 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 same conditions. Nevertheless,
they were able to grow on
glucose-peptone agar with 0.5% Tweens
40 and 60, a result which
differed from the identification of
this species proposed by
Guého et al. (
12). According to these
findings, these
lipid-dependent species had the same characteristics
as the species
M. obtusa, except for their growth on glucose-peptone
agar
with 0.5% Tweens 40 and
60.
Discussion.
The importance of M. pachydermatis in
dogs has been extensively reported (13, 16). This species
can play an important role in chronic dermatitis and otitis externa in
carnivores, especially in dogs. This yeast has an opportunistic nature,
and it may become pathogenic with any alteration in the skin surface
microclimate or in host defense. Canine otitis externa and seborrheic
dermatitis are frequently associated with large numbers of M. pachydermatis (22). The frequency of isolation of this
species from the external ear canal is reported to be 15 to 49% for
healthy dogs and may increase up to 50 to 83% for dogs with otitis
externa (10).
Classically, lipid-dependent species were related to human skin only,
but it is now known that the skin of different animals
also can be
colonized by lipid-dependent species in addition to
M. pachydermatis (
14).
M. slooffiae seems to
represent a large
proportion of the cutaneous yeast flora in pigs, and
it has been
isolated from normal skin in sheep and goats as well
(
12). The
following lipid-dependent species have been
isolated from healthy
bovines (
8,
12):
M. globosa,
M. furfur,
M. slooffiae,
M. obtusa, and
M. sympodialis. The presence of
lipid-dependent species
in carnivores has been recently demonstrated.
M. sympodialis (
3),
M. globosa
(
4), and
M. furfur (
6) have been
described to
colonize the skin and mucosae of healthy cats.
M. furfur and
M. sympodialis also have been reported from
canine specimens tested
by identification techniques such as the
assimilation of Cremophor
EL, the splitting of esculin, and pigment
synthesis (
21).
The role of lipid-dependent species in human skin is well documented.
They are commensal yeasts that may become etiological
agents of
cutaneous and systemic diseases. However, very little
is known about
their role in nonhuman animal skin. A skin disorder
in goats associated
with
Malassezia cells morphologically different
from
M. pachydermatis cells was described on the basis of
histological
techniques, but the isolation in vitro of the casual agent
was
unsuccessful (
2). More recently, lipid-dependent species
have
been isolated in bovines with otitis externa (
M. globosa,
M. slooffiae,
M. furfur, and
M. sympodialis) (
8), and two cases of feline
otitis externa associated with
M. sympodialis have been
reported
from our laboratory (
7).
In this study, three atypical lipid-dependent yeasts have been
isolated. 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 mechanism similar
to
that of
M. pachydermatis in the skin. They are members of
the
cutaneous flora of some animals, and they might become pathogenic
under the same predisposing factors. However, more studies would
be
required to determine their role in animal skin. For this reason,
culture media with lipid sources, such as SGA supplemented with
olive
oil, Leeming's medium, or modified Dixon's agar, should
be used in
addition to media without lipid sources. Our finding
of lipid-dependent
isolates indicates that canine otitis externa
can be associated with
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 polyoxyethylene
sorbitan
esters (Tweens 20, 40, 60, and 80), the Cremophor EL
assimilation test,
and the splitting of esculin was not able to
identify our canine
lipid-dependent isolates. Some differences
in the assimilation patterns
were observed on comparison with
the type strain patterns. It is likely
that the number of lipid-dependent
species that do not fit the
described type species profiles increases
as long as further isolates
are obtained from different hosts.
In these such situations,
complementary tests, including molecular
techniques, would be required
to differentiate atypical lipid-dependent
isolates. On the other hand,
the possibility of some variations
in assimilation patterns within a
species should be
considered.
| |
ACKNOWLEDGMENTS |
We thank Autonomous University of Barcelona Veterinary Teaching
Hospital and various veterinary clinics from the Barcelona region for
the samples kindly provided for this investigation.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Departament de
Patologia i Producció Animals (Microbiologia), Facultat de
Veterinària, Edifici V, Room V0-285, Universitat Autònoma
de Barcelona, 08193-Bellaterra (Barcelona), Spain. Phone: 34 93 581 17 49. Fax: 34 93 581 20 06. E-mail: javier.cabanes{at}uab.es.
| |
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Journal of Clinical Microbiology, June 2000, p. 2383-2385, Vol. 38, No. 6
0095-1137/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
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