Genetic Relationship between Human and
Animal Isolates of Candida albicans
Anke Edelmann, Monika Krüger and Jan Schmid
J. Clin. Microbiol. 2005, 43(12):6164. DOI:
10.1128/JCM.43.12.6164-6166.2005.
These include:
REFERENCES
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JOURNAL OF CLINICAL MICROBIOLOGY, Dec. 2005, p. 6164–6166
0095-1137/05/$08.00⫹0 doi:10.1128/JCM.43.12.6164–6166.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.
Vol. 43, No. 12
Genetic Relationship between Human and Animal Isolates
of Candida albicans
Anke Edelmann,1* Monika Krüger,2 and Jan Schmid3
Institute of Biochemistry, Department of Molecular Biochemistry, Medical Faculty, University of Leipzig, Leipzig, Germany1;
Institute of Bacteriology and Mycology, Veterinary Faculty, University of Leipzig, Leipzig, Germany2;
and Institute of BioSciences, Massey University, Palmerston North, New Zealand3
Received 24 June 2005/Returned for modification 31 July 2005/Accepted 4 October 2005
Candida albicans can be found in the intestinal tracts and in
the oral cavities of healthy individuals, and is also the predominant causative agent of human candidosis (5, 12, 17). In addition, all domestic animals like cattle, horses, pigs, cats, and
dogs as well as birds are susceptible to Candida infections (4).
This suggests that animals could be vectors of transmission or
reservoirs of strains causing human disease and may present a
risk for immunocompromised patients. Although, many case
reports of candidiasis in animals are published (6, 9, 11), very
little is known about the identity and origins of these infecting
strains and the genetic relationship among C. albicans isolates
from human and animal sources (1).
To investigate whether C. albicans strains from humans
are genetically distinct from animal isolates, 27 strains isolated from sputum, lungs, or feces of humans with candidemia (16 from human immunodeficiency virus-positive patients and 11 from other patients) were analyzed by Ca3
fingerprinting (13). In addition, 18 isolates from various
animal species recovered from specimens submitted by veterinarians in Saxony (Germany) to the Institute of Bacteriology and Mycology of the Veterinary Faculty were typed as
well (see Table 1 for details of isolates). None of the humans
were owners of, or in close contact with, the animals sampled.
C. albicans strains were grown in YPD medium (1% yeast
extract, 2% Bacto peptone, 2% glucose) at 30°C and 250 rpm
overnight. For Southern blot analysis, genomic DNA was extracted as described previously (3) and digested with EcoRI.
Blots were hybridized with probe Ca3 (13) labeled with digoxigenin using a DIG DNA labeling and detecting kit (Roche).
After prehybridization and hybridization performed at 68°C,
the membrane was washed twice with 2⫻ SSC (1⫻ SSC is 0.15
M NaCl plus 0.015 M sodium citrate, pH 7.0)-0.1% sodium
dodecyl sulfate (SDS) at 25°C for 5 min and twice with 1⫻
SSC-0.1% SDS at 68°C for 15 min. The immunological detection was carried out as recommended by the manufacturer. To
quantify strain differences, molecular size and intensity of
bands from C. albicans isolates were scored by comparison
with the pattern of the reference strain 3153A on the same blot
according to Schmid et al. (13). Signals of low intensity ⱕ2.1 kb
and fast-evolving high-molecular-size bands ⱖ10.3 kb (10)
were not included in the comparison. Intensity of hybridization
was defined in arbitrary units: 0 U, absence of a band, 1 U,
weak, 2 U, medium, and 3 U, strong signal (13). Paupⴱ
TABLE 1. Sources of C. albicans isolates used in this study
Strain
Site(s) of
isolation
Human samples
H1
H2 b
H3
H4 b
H7–H9 b
H10
H11–H22 b
H23–H27
H35
H40
H42
* Corresponding author. Mailing address: Institut für Biochemie, Abt.
Molekulare Biochemie, Medizinische Fakultät, Universität Leipzig,
Johannisallee 30, 04103 Leipzig, Germany. Phone: 49-341-9722156. Fax:
49-341-9722159. E-mail: [email protected].
Feces
Feces
Feces
Feces
Skin
Rectal swab
Feces
Feces
Rectal swab
Feces
Guinea pig
Ferret
Giraffe
Alpaca
Baikal seal c
Magpie
Peacock
Turtle c
Feces
Feces
Intestine
Feces
Feces, mouth
Feces
Feces
Feces, mouth
b
c
6164
Leipzig
Leipzig
Leipzig
Leipzig
Leipzig
Leipzig
Leipzig
Leipzig
Leipzig
Leipzig
Leipzig
Animal samples
Cat-1
Cat-2
Dog-1
Dog-2
Dog-3
Horse-1
Horse-2
Pig
Rabbit
Chicken
a
Sample origin
(Germany) (owner)
Saxony a
Thuringia
Saxony a
Thuringia
Thuringia
Saxony a
Groitzsch
Lüneburg a
Saxony a
Möckern
(poultry farm)
Gross-Glienicke
Saxony a
Leipzig (zoo)
Saxony a
Leipzig (zoo)
Engelsdorf
Leipzig (zoo)
Saxony a
Private owner.
Human immunodeficiency virus status of all patients was known.
Animal with established candidemia.
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Analyzing Candida albicans isolates from different human and animal individuals by Ca3 fingerprinting, we
obtained no evidence for host-specific genotypes and for the existence of species-specific lineages, even though
a certain degree of separation between human and animal isolates was found. Therefore, animals could
potentially serve as reservoirs for human Candida infection.
VOL. 43, 2005
NOTES
6165
(Sinauer) was used to generate mean character distances between strains, based on these patterns, and to display relationships between strains as a neighbor-joining tree.
For each strain a unique band pattern was obtained (Fig. 1).
Results from Ca3 fingerprint analyses were processed, and
strain relations are depicted in a neighbor-joining tree (Fig. 2).
The distribution of the three canine, the two feline, and the
two horse isolates throughout the tree argued against a monophyletic origin of isolates found on the same host species. The
tree suggested some degree of separation of human from animal isolates because it showed apparent small groups of
closely related isolates which contained either only human
(e.g., H11, H4, H14, H2) or animal (giraffe, horse-2, magpie)
isolates. To test for genetic separation between human and
animal isolates, a nearest-neighbor analysis (2) was carried out
as described in detail recently (14): We calculated how often a
given human isolate would have an animal isolate as its closest
relative, assuming that no separation between human and animal isolates exists. In this case the probability that a human
isolate has an animal isolate as its closest relative would be
determined solely by the ratio of the number of animal strains
to the sum of all remaining human plus animal isolates (18/44).
By multiplying this ratio (0.409) with the number of human
isolates typed (n ⫽ 27), we determined that in 11 cases one
would expect a human isolate to have an animal isolate as its
closest relative, if there is no genetic separation. Using the
matrix of Ca3 fingerprint-based distances, we then determined
how often human isolates were indeed the closest relative to
animal isolates. This was the case for only four human isolates
(pairs of isolates are marked in Fig. 2). The binominal probability
(16) of finding four or fewer human isolates with an animal isolate
as their closest relative when 11 such cases are expected under the
assumption of lack of genetic separation was 0.0037. Thus, our
data indicated genetic separation between animal and human
isolates. There were two additional cases in which a human isolate
was equally close to animal and human isolates, marked in Fig. 2
FIG. 2. Genetic relations of Candia albicans isolates from humans and animals. A neighbor-joining tree was calculated from Ca3
fingerprint results for 45 C. albicans isolates from humans (H1 to
H42) and from various animal species. Curved arrows indicate cases
where an animal isolate was the closest relative to a human isolate.
Two groups of isolates are also marked in which a human isolate
was as close to an animal isolate as to other human isolates (triangles, H7 plus its closest isolates: chicken, H24, and H42; circles, H12
plus its closest isolates: rabbit, H2, and H14). To assess the phylogenetic support for groupings on the tree, we performed a bootstrap
resampling analysis. Bootstrap support values higher than 50% are
indicated next to the appropriate nodes.
(closest isolates of H7: chicken, H24, and H42; closest isolates of
H12: rabbit, H2, and H14). Even if we treated these as additional
instances in which an animal isolate was the closest relative of a
human isolate, the binominal probability of finding six or fewer of
such cases in the absence of genetic separation would be still only
0.0357.
It is noteworthy that, in the above-mentioned six cases,
where human isolates were closest to animal isolates, the average genetic distance between these pairs (0.239 ⫾ 0.077;
including H7 and H12, which are equally close to other human
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FIG. 1. Ca3 fingerprints of Candia albicans isolates. EcoRI-digested genomic DNA from eight animal and human C. albicans isolates was subjected to Southern blot analysis applying a digoxigeninlabeled Ca3 sequence. DNA from C. albicans strain 3153A (13) was
included as a molecular weight and band intensity standard.
6166
NOTES
We thank P. Nenoff, Department of Dermatology, University of
Leipzig, Germany, for kindly providing the human C. albicans strains.
And we are grateful to Torsten Schöneberg and Wolfgang Schellenberger for critical reading of the manuscript.
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and animal isolates) was not significantly larger than that between the closest related pairs of human isolates (0.208 ⫾
0.086) (P ⬍ 0.44, t test). Thus isolates from animal species can
be as closely related to human strains as closely related human
isolates are to each other.
One plausible explanation for the observed genetic separation in the absence of evidence for species-specific monophyletic groups is that genetic separation is due to barriers to
transmission (low frequency of contact). In other words, restriction of human-animal transmission and genetic drift may
account for the presence of different genotypes of human and
animal C. albicans strains in our sample. An alternative or
additional factor could be that, while there is no absolute
species specificity, different C. albicans clades may differ in the
frequency in which they colonize various species. The fact that
for many of the animal isolates pathogenic significance was not
established, whereas all human isolates were disease causing
(Table 1), should only have a small impact on our results: there
is no evidence for genetic separation between human C. albicans strains that act as pathogens and strains that act as commensals (15). However, particular genotypes can be overrepresented among pathogenic human isolates (13, 14), and thus
our human isolates may not be fully representative of the
genetic diversity of human isolates in the area. Nevertheless,
the reliability of the analysis is supported by the fact that the
relationships among human isolates found in this work are
consistent with the diversity and phylogenetic structure of human isolates assessed previously (7, 8, 15).
In summary, our phylogenetic analysis of C. albicans isolates
from different animal and human sources did not reveal the
existence of species-specific lineages, even though the nearestneighbor analysis revealed some degree of separation between
human and animal isolates. While barriers to transmission
between animals and humans may in some cases be higher than
between humans and humans, our data do not suggest that
they are insurmountable. Further research on the relationship
of isolates from patients and their companion animals is necessary to obtain an estimate of the frequency of animal-humananimal transmission. However, our study and a previous study
(1) indicate that animals have to be considered as potential
sources of Candida infections of human individuals especially
when humans are immunodeficient.
J. CLIN. MICROBIOL.