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Institute of Biochemistry, Department of Molecular Biochemistry, Medical Faculty, University of Leipzig, Leipzig, Germany
Institute of Bacteriology and Mycology, Veterinary Faculty, University of Leipzig, Leipzig, Germany
Institute of BioSciences, Massey University, Palmerston North, New Zealand
ABSTRACT
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.
TEXT
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 2x SSC (1x 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 1x 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 2.1 kb and fast-evolving high-molecular-size bands 10.3 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). Paup (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 (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 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 nearest-neighbor 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-human-animal 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.
ACKNOWLEDGMENTS
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 Schneberg and Wolfgang Schellenberger for critical reading of the manuscript.
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