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Journal of Clinical Microbiology, September 2001, p. 3382-3385, Vol. 39, No. 9
0095-1137/01/$04.00+0 DOI: 10.1128/JCM.39.9.3382-3385.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Application of PCR to Distinguish Common Species of
Dermatophytes
Elisabetta
Faggi,1,*
Gabriella
Pini,1
Enza
Campisi,1
Chiara
Bertellini,1
Elisa
Difonzo,2 and
Francesca
Mancianti3
Dipartimento di Sanità
Pubblica
Sezione Microbiologia, Università di Firenze, 50134 Florence,1 Dipartimento di Scienze
Dermatologiche, Università di Firenze, 50121 Florence,2 and Dipartimento di
Patologia Animale, Università di Pisa, 56124 Pisa,3 Italy
Received 30 March 2001/Returned for modification 16 May
2001/Accepted 8 July 2001
 |
ABSTRACT |
This report describes the application of PCR fingerprinting for the
identification of species and varieties of common dermatophytes and
related fungi utilizing as a single primer the simple repetitive oligonucleotide (GACA)4. The primer was able to amplify all
the strains, producing species-specific profiles for Microsporum
canis, Microsporum gypseum, Trichophyton
rubrum, Trichophyton ajelloi, and
Epidermophyton floccosum. Intraspecific variability
was not observed for these species. Instead, three different profiles were observed in the Trichophyton mentagrophytes group.
| |
TEXT |
Routine procedures for dermatophyte
species identification rely on examination of the colony (pigmentation
of the surface and reverse sides, topography, texture, and rate of
growth) and microscopic morphology (size and shape of macroconidia and
microconidia, spirals, nodular organs, and pectinate branches). Further
identification characteristics include nutritional requirements
(vitamins and amino acids) and temperature tolerance, as well as urease
production, alkaline production of bromocresol purple medium, in vitro
hair perforation, etc. (9, 16). Morphological and
physiological characteristics can frequently vary; in fact, the
phenotypic features can be easily influenced by outside factors such as
temperature variation, medium, and chemotherapy (11) and
therefore strain identification is often difficult.
In the last few years genotypic approaches have proven to be useful for
solving taxonomic problems regarding dermatophytes; in fact, genotypic
differences are considered more stable and more precise than phenotypic
characteristics (2, 11).
Molecular methods, such as restriction fragment length polymorphism
analysis of mitochondrial DNA (1, 7, 8), sequencing of the
internal transcribed spacer (ITS) region of the ribosomal DNA (3,
4), sequencing of protein-encoding genes (5, 6),
and PCR (random amplification of polymorphic DNA [RAPD] [15], arbitrarily primed PCR [AP-PCR] [10,
11], and PCR fingerprinting [2]), have brought
important progress in distinguishing between species and strains.
However, most of these techniques (e.g., restriction fragment length
polymorphism analysis, sequencing) are complex, laborious,
time-consuming, and not easily employable for routine identification of
dermatophytes; in contrast, PCR technology is simple, rapid, and, in
the absence of specific nucleotide sequence information for the many
dermatophyte species, able to generate species-specific or
strain-specific DNA polymorphisms on the basis of characteristic band
patterns detected by agarose gel electrophoresis (2, 11).
This report describes the application of PCR fingerprinting for the
identification of species and varieties of common dermatophytes and
related fungi utilizing as a single primer the simple repetitive oligonucleotide (GACA)4 previously used by Meyer
and others to distinguish strains of Cryptococcus neoformans
(12, 13) and species of the genus Candida
(14).
The species and varieties we have studied are Microsporum
canis, Microsporum gypseum, Trichophyton
ajelloi, Trichophyton mentagrophytes var.
asteroides, T. mentagrophytes var.
granulosum, T. mentagrophytes var.
lacticolor, T. mentagrophytes var.
radians, T. mentagrophytes of undetermined
variety, Trichophyton interdigitale,
Trichophyton rubrum, and Epidermophyton
floccosum.
Furthermore, the study was conducted on various strains both from
collections and from clinical isolation with the aim of finding the
presence of an intraspecific variability.
Strains.
Out of a total of 140 strains selected for the study,
29 were obtained from the collection of the Institut Pasteur of Paris, France. One hundred eleven clinical isolates were recovered from humans
with dermatophytosis as well as from cats and dogs with or without
visible lesions. The clinical strains were isolated in Florence
(Department of Public HealthMicrobiology Unit and Department of
Dermatological Sciences) and Pisa (Department of Animal Pathology),
Italy, during 1997 and 1998 and identified using conventional culture
and microscopic techniques.
The origins of the strains are listed in Tables
1 and 2.
DNA extraction.
The strains were grown in Sabouraud's
dextrose agar at 25°C; after 2 weeks some mycelium was cut from the
agar and transferred to Sabouraud's dextrose broth. After 2 weeks at
25°C, superficial mycelial growth was transferred to a mortar, washed
with distilled water, and pestled. For rapid DNA extraction we
used the Dynabeads DNA Direct System I (Dynal) based on
biomagnetic separation.
In brief, about 20 µl of pestled mycelium was transferred to an
Eppendorf tube and incubated with 200 µl of Dynabeads (paramagnetic
polystyrene beads in lysis buffer) for 10 min at 65°C so as to
obtain
cell lysis and the adsorption of the released DNA to the
Dynabead
surface. This step was followed by magnetic separation
of the
DNA-Dynabeads complex and by two or three subsequent washings
that
removed any residual contaminant and eliminated potential
PCR
inhibitors. The DNA-Dynabeads complex was resuspended in TE
buffer (10 mM Tris-HCl [pH 8], 1 mM EDTA), and DNA was eluted
for 5 min at
65°C; it was then ready for PCR or storage at
20°C.
For some strains several cultures and extractions were
performed.
PCR fingerprinting.
The simple repeat sequence
(GACA)4 was used as a single primer
(12-14) in the PCR amplification.
Amplification reactions were performed in volumes of 50 µl
containing 25 ng of template DNA, reaction buffer (10 mM Tris-HCl
[pH 8.3], 50 mM KCl), 2.5 mM MgCl
2, 200 µM (each) dATP, dCTP,
dGTP, and dTTP, 160 ng of primer, and 2.5 U of
Taq DNA polymerase
(GeneAmp PCR Core reagents;
Perkin-Elmer Cetus, Norwalk, Conn.).
The samples were overlaid with sterile paraffin oil (Carlo Erba) and
PCR was performed for 39 cycles in a DNA Thermal Cycler
(Perkin-Elmer
Cetus) with 1 min of denaturation at 93°C, 1 min
of annealing at
50°C, and 1 min of extension at 72°C and then
a final extension for
7 min at 72°C.
PCR products (20 µl/sample) were separated by electrophoresis in 1%
agarose gels for 2 h at 5V/cm in 0.5× TBE buffer (0.045
M
Tris-borate [pH 8.3], 1 mM EDTA). Amplification products were
detected by staining with ethidium bromide and were visualized
under UV
light.
Each sample of genomic DNA was amplified in duplicate in the
same PCR and in repeated PCRs at different
times.
The genomic analysis of all the strains was done utilizing
rigorously standardized concentrations of the reagents, the same
thermal cycler, and the same cycling
conditions.
The primer we used [(GACA)
4] was able to
amplify all the strains, and the method used was shown to be a simple,
rapid, and
reproducible technique. In fact, multiple extractions of the
same
strain starting with cultures grown for different times
produced
PCR fingerprinting profiles showing the same distribution of
bands
of both strong and weak
intensity.
Each strain produced the same genomic profiles whether in the
same PCR (double amplified sample) or in PCRs repeated at different
times. Occasional changes in band intensities were observed which
could
be attributed to slight variations in the reaction conditions
(
13).
To confirm that the observed bands were really amplified
genomic DNA and not primer artifacts, genomic DNA was
omitted from
the control reaction mixture for each PCR. We did not
observe
amplification products in any control
reaction.
The PCR fingerprints of all strains yielded up to 11 bands, ranging
from approximately 394 to 2,399 bp in length; the number
of brightly
colored fragments varied from 2 to 5 according to
the species, while
that of the weakly colored ones varied from
2 to
8.
All 53 strains of
M. canis produced profiles which are
perfectly superimposable without distinction between the collection
strains and the strains isolated from humans, cats, and dogs,
with or
without the presence of clinical lesions (Fig.
1).
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FIG. 1.
PCR fingerprints of M. canis strains
(each strain was amplified in duplicate). Lanes: M, molecular weight
marker VI (Boehringer Mannheim), size range, 154 to 2,176 bp; 1, control reaction without template DNA; 2 to 7, three strains from
humans; 8 to 15, four strains from dogs; 16 to 19, two strains from
cats.
|
|
Species-specific profiles were also observed for the species
M. gypseum (16 strains),
T. rubrum (14 strains),
T. ajelloi (5
strains), and
E. floccosum (9 strains) for
which no intraspecific
variability was noted; the complexity of the
profiles of
M. canis,
M. gypseum, and
E. floccosum was contrasted by the simplicity
of those presented by
T. rubrum and
T. ajelloi, which for their
limited
number of bands and their distribution are easily distinguished
from
those of the other species examined (Fig.
2).
View larger version (53K):
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FIG. 2.
PCR fingerprints of five different dermatophyte species
(two different strains from each species). Lanes: M, molecular weight
marker VI (Boehringer Mannheim), size range, 154 to 2,176 bp; 1 and 2, M. canis; 3 and 4, M. gypseum; 5 and 6, T. rubrum; 7 and 8, T. ajelloi; 9 and 10, E. floccosum.
|
|
The profiles of
T. mentagrophytes and
T. interdigitale were also complex and we noted in them a discrete
variability which
allowed us to group the strains examined into three
different
profiles. The first and second profiles differ by only a
fragment
(approximately 653 bp) while the third profile is very
different
from the other two (Fig.
3).
View larger version (58K):
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[in a new window]
|
FIG. 3.
PCR fingerprints showing three different profiles of
T. mentagrophytes group strains (each strain amplified
in duplicate). Lanes: M, molecular weight marker VI (Boehringer
Mannheim), size range, 154 to 2,176 bp; 1 and 2, T.
mentagrophytes var. granulosum; 3 to 6, T. interdigitale (two strains); 7 and 8, T.
mentagrophytes var. asteroides; 9 and 10, T. mentagrophytes var. radians; 11 and
12, T. mentagrophytes var. lacticolor; 13 and 14, T. mentagrophytes of unspecified variety.
|
|
In the first profile we find all the strains of
T. mentagrophytes var.
granulosum and of
T. interdigitale, in the second
T. mentagrophytes var.
asteroides and
T. mentagrophytes var.
radians,
and in the third
T. mentagrophytes var.
lacticolor; the strains
of
T. mentagrophytes of
unspecified variety are distributed as
follows in all three profiles:
in the first we find strains isolated
from both humans and animals and
in the second and third we find
only strains isolated from humans
(Table
3).
PCR fingerprinting has proven to be a simple and reproducible method.
In fact, by strictly maintaining the experimental conditions
we have
had superimposable
profiles.
PCR fingerprinting, along with a fast DNA extraction method,
proved to be a rapid method in comparison to other techniques
of
molecular biology (restriction fragment length polymorphism
analysis,
sequencing of the ITS region, and sequencing of protein-encoding
genes).
In fact, in about 6 h we can obtain electrophoretic profiles
starting from cultures, as the DNA extraction technique we used
does
not require more than half an hour and the amplification
requires about
5
h.
The primer we used produced species-specific profiles for
M. canis,
M. gypseum,
T. rubrum,
T. ajelloi, and
E. floccosum and
allowed us to detect
three different profiles for
T. mentagrophytes in
relation to the varieties studied. The fact that all the strains
of
T. interdigitale belong to one of these profiles confirms
the
notion that this species is closely related to or a variety of
T. mentagrophytes.
The great diversity of profiles between the
T. mentagrophytes group and
T. rubrum seems particularly
interesting and makes
the two species quite distinguishable, while with
the classical
technique their identification is often
difficult.
These species-specific profiles could be used for identifying strains
that do not present typical morphological characteristics
and therefore
cannot be identified in the classical
way.
In our experience we isolated some strains from dogs and cats that did
not produce conidia but presented a characteristic
orange pigmentation
on the reverse side of the colony. PCR fingerprinting
of these strains
has produced electrophoretic profiles that are
superimposable with
those produced by the strains of
M. canis reported in this
work, causing us to hypothesize that they belong
to this species
(unpublished
data).
Dermatophytes in culture easily lose their typical morphological
characteristics, so PCR fingerprinting could be used for
reidentifying
the collection strains. In our experience we were
able to reidentify
numerous strains of our collection which had
lost their typical
morphological characteristics (unpublished
data).
Since genomic research does not necessarily imply the use of
live organisms, PCR fingerprinting could also be used for studying
dead
strains. We have no experience in this regard but Liu et
al.
(
11) have been able to confirm the identities of
20-year-old
nonviable dermatophyte isolates by AP-PCR.
In conclusion, while the only disadvantage of the use of PCR
fingerprinting for identifying dermatophytes is the relatively
higher
cost in comparison to the classical method, the advantages
of its use
are
many.
It is a technique of simple execution, it is rapid in comparison with
other techniques of molecular biology, especially thanks
to the speed
of the DNA extraction, and it can be applied in cases
where it is
necessary to identify strains not presenting typical
morphological
characteristics.
Therefore, this method can be of great utility when it is not possible
to use, for the above-specified reasons, the classical
method, which is
still valid and advisable for identifying strains
with
well-characterized morphological
aspects.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Dipartimento di
Sanità PubblicaSezione Microbiologia, Viale Morgagni 48, 50134 Florence, Italy. Phone: (055) 411081. Fax: (055) 4223895. E-mail:
efaggi{at}unifi.it.
| |
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Journal of Clinical Microbiology, September 2001, p. 3382-3385, Vol. 39, No. 9
0095-1137/01/$04.00+0 DOI: 10.1128/JCM.39.9.3382-3385.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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