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Journal of Clinical Microbiology, November 1999, p. 3586-3589, Vol. 37, No. 11
Service de Microbiologie,
Received 22 March 1999/Returned for modification 10 June
1999/Accepted 28 July 1999
Fusarium spp. have emerged as major opportunistic
fungal agents. Since new antifungal agents exhibit variable activity
against Fusarium isolates depending on the species, rapid
identification at the species level is required. Conventional culture
methods are difficult, fastidious, and sometimes inconclusive. In this work, we sequenced a 440-bp fragment encoding the 28S rRNA from 33 Fusarium isolates belonging to six Fusarium
species associated with human infections. The data were then analyzed
by the neighbor-joining method. By using distance matrix analysis and
constructing the phylogram, we could easily distinguish the different
species for all but one isolate. The method also allowed
differentiation between the closely related genera
Acremonium and Cylindrocarpon. In contrast to
the case with conventional methods, the results could be obtained within 48 h from a 3-day culture and are independent of mycologist experience, making this method rapid and reliable for identification of
Fusarium species isolated from patients.
The fungi belonging to the genus
Fusarium are well-known plant pathogens and food
contaminants that also cause superficial and subcutaneous infections,
such as onychomycosis and keratomycosis, in humans (9). They
have recently emerged as major opportunistic agents in
immunocompromised hosts, especially in patients with hemopathy (2,
7). They are now considered the third most common fungal genus
(after Candida and Aspergillus) isolated from systemic infections in bone marrow transplantation patients
(8). Four species account for more than 95% of human
infections: Fusarium solani, Fusarium moniliforme
(Fusarium verticilloides), and Fusarium oxysporum
are each responsible for about 30% of the cases, whereas Fusarium dimerum is involved in 5% of the cases (6,
7). Diagnosis of Fusarium at the species level is
based on conventional methods, which include the description of
colonies on appropriate media (texture, color, and pigment etc.) and
microscopic description of conidiogenous cells and conidia. This can be
best observed after 2 weeks of incubation, lengthening the time for a
definitive diagnosis. Because of important variations of characters,
such as pigmentation and growth rate, that are often seen within a given species, only well-trained mycologists are able to ensure the
diagnosis (6). The results are thus frequently inconclusive, as seen in various reports where one-third to one-half of the isolates
are not identified at the species level (2, 7). Identification at the species level is important for epidemiological purposes and may become absolutely necessary because some new antifungal agents exhibit variable activity against Fusarium
depending on the species (1, 13). In this work, we report a
rapid method for the identification of significant Fusarium
species involved in human infections by using rRNA gene (rDNA) sequencing.
Strains.
The fungal isolates used in this work are listed in
Table 1. A total of 33 isolates belonging
to six different Fusarium species were tested, including 18 reference strains and 15 clinical isolates collected from superficial
and deep infections. Three clinical isolates and one reference strain
belonging to the genus Acremonium and two
Cylindrocarpon tonkinense reference strains were also studied due to the difficulties in distinguishing between these genera
and Fusarium (6). In addition, nine control
isolates from unrelated fungi were included to test the specificity of the designed primers (see below). The identification of
Fusarium and Acremonium isolates was performed
according to the colonial aspects and microscopic morphology after 7 and 14 days of culture at 25°C on potato dextrose agar (Difco
Laboratories, Detroit, Mich.) plates in daylight. Examination of
micro-, meso-, and macroconidia, chlamydospores, and phiallides allowed
differentiation between the Fusarium species (4,
6). All of the isolates were stored in 10% glycerol at DNA extraction.
After a 3-day incubation at 35°C on
Sabouraud agar (Sanofi-Diagnostic Pasteur, Marnes la Coquette, France)
plates, cultures were discharged in 300 µl of distilled water in a
microcentrifuge tube. A volume of 100 µl of Chelex solution (10%
[wt/vol] Chelex-100 [Bio-Rad, Hercules, Calif.] in an aqueous
solution of 0.1% [wt/vol] sodium dodecyl sulfate sodium salt
[Sigma, St. Louis, Mo.], 1% [vol/vol] Nonidet P-40 [Sigma], and
1% [vol/vol] Tween 80 [Sigma]) was added. The tubes were incubated
at 95°C for 30 min and then on ice for 5 min. DNA was removed from
the supernatant after 5 min of centrifugation (10,000 × g)
and stored at Amplification and sequencing.
Two oligonucleotides (Fus1
[5'-TGAAATCTGGCTCTCGGG] and Fus2
[5'-CATGCGCGAACCTCAGTC]) were designed after comparison of
Fusarium 28S rDNA sequences in the GenBank database.
Amplification was performed in a volume of 50 µl with 10 mM Tris-HCl,
50 mM KCl, 2.5 mM MgCl2, 0.01% (wt/vol) gelatin, 5 µl of
DNA, a 0.25 µM concentration of each oligonucleotide primer, 0.2 mM
deoxynucleoside triphosphates (Pharmacia, Uppsala, Sweden), 0.3 U of
Taq Gold (PE Applied Biosystems, Foster City, Calif.), and 5 µl of DNA. Thermal cycling parameters were as follows: 10 min at
94°C, followed by 35 cycles of 30 s at 94°C, 90 s at
64°C, and 90 s at 72°C and a final extension of 10 min at
72°C. The PCR products were separated by electrophoresis and
visualized under UV. Amplification products were purified by using
microspin S-400 HR purification columns (Pharmacia). The sequence
reactions were made with 5 µl of purified amplified DNA, 4 pmol of
primers, and 4 µl of Big Dye Terminator (PE Applied Biosystems)
according to the manufacturer's instructions. The extension products
were then precipitated, washed, and resuspended in Template Suppression
Reagent (PE Applied Biosystems). After denaturation at 95°C for 2 min, the samples were loaded onto POP6 capillary columns in a ABI Prism
310 Genetic Analyzer (PE Applied Biosystems). Sequences of both strands
were determined.
Sequence alignments and phylogenetic trees.
Sequences were
edited with the Sequence Navigator software (PE Applied Biosystems).
Multiple sequence alignments were performed with the Clustal X version
1.64 software (Higgins, Heidelberg, Germany). Trees and matrix
distances were constructed with the Phylip package 3.572 by using the
neighbor-joining method. The degree of confidence in phylogenetic
branching was assessed by using 1,000 bootstrap resamplings.
Nucleotide sequence accession numbers.
The nucleotide
sequences of the strains and isolates tested in this work appear in the
GenBank nucleotide sequence database under the accession numbers shown
in Table 1.
In this work, we compared the conventional methods for
identification of Fusarium species with a 28S rDNA sequence
method. Using the newly designed primers Fus1 and Fus2, we were able to amplify a 480-bp fragment from 33 Fusarium strains and
isolates and from strains and isolates from the closely related genera Acremonium and Cylindrocarpon. In contrast, we
failed to amplify the DNAs from nine reference strains belonging to the
unrelated species listed in Table 1. Sequencing of the PCR products led to 10 sequence classes for Fusarium strains and isolates,
each containing 1 to 10 isolates. Comparison with Fusarium
sequences from the GenBank database demonstrates complete interspecies
identity with those of Fusarium semitectum (accession no.
X80813), F. oxysporum (U34537 and U34542), and F. moniliforme (U34528). The sequence of F. dimerum
(U88105) exhibits close similarity with that of F. dimerum
sequence class 2, with a difference of only 6 nucleotides (98.6%
homology). Comparison of sequences for F. solani is possible
only with sequences obtained from F. solani forma speciales
phaseoli isolates (L36629, L36630, L36632, and L36634),
which are involved in the soybean sudden death syndrome. They exhibit a
closer homology with C. tonkinense sequence class 2 (98.16%) than with F. solani sequence class 1 (96.74%). Multiple alignment of our sequence classes was carried out by using
Clustal X software (15). These data were used to construct an unrooted phylogenetic tree by the neighbor-joining method
(12). As illustrated in Fig.
1, each Fusarium species as
determined by conventional methods formed a distinct clade, with a
minimum bootstrap confidence value of 71% for F. solani
(Fig. 1). Interspecies homologies for Fusarium never reached
higher than 99.01% (i.e., for Fusarium chlamydosporum and
F. semitectum) (Fig. 2). In
contrast, with the exception of F. dimerum (discussed
below), intraspecies homologies ranged from 99.09% for F. solani to 100% for F. oxysporum. An isolate thus can
be readily assigned to one species when its homology with one of the
sequence classes is higher than 99.1%.
0095-1137/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Identification of Fusarium Species
Involved in Human Infections by 28S rRNA Gene Sequencing
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results and Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results and Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results and Discussion
References
80°C
until tested.
TABLE 1.
Strains and isolates tested for 28S rDNA sequences in
this study
80°C until used.
RESULTS AND DISCUSSION
Top
Abstract
Introduction
Materials and Methods
Results and Discussion
References
View larger version (14K):
[in a new window]
FIG. 1.
Unrooted phylogenetic tree derived from 28S rDNA
sequences of Fusarium spp., C. tonkinense,
and Acremonium spp. The tree was constructed by using the
neighbor-joining method. Bar, 1% estimated sequence divergence. cl,
class.
View larger version (27K):
[in a new window]
FIG. 2.
Homology between Fusarium spp.,
Acremonium spp., and C. tonkinense, derived from
28S rDNA sequences. The sequence classes (cl) correspond to those in
Table 1.
The isolate IHEM 5322, considered F. dimerum, exhibited a unique sequence, with 8.25% divergence from the other sequence class containing three strains of F. dimerum. Interestingly, this isolate had deletions at positions 398 and 403, a characteristic shared only with the other sequence class corresponding to F. dimerum. We have no explanation for this discrepancy. (It should be mentioned that this strain had been collected not from a patient but from the recycled water of a spray humidifier.) Such a discrepancy between a molecular method (in this case using species-specific probes) and the conventional method has already been described for a Fusarium reference strain (3), suggesting that conventional methods may not be accurate for some strains with atypical phenotypic characteristics.
While the aim of this work was not a phylogenic reevaluation, we noted that there are closer relationships between some genera tested in this study than between some species within the Fusarium genus. This is illustrated by the grouping of F. solani, Acremonium spp., and C. tonkinense (intersequence class divergences lower than 6.24%), whereas F. moniliforme and F. oxysporum exhibited mean divergences from F. solani between 6.98 and 7.42%, respectively. Interestingly, F. solani, Acremonium spp., and Cylindrocarpon are characterized by teleomorphs belonging to the genus Nectria, whereas F. moniliforme and F. oxysporum have teleomorphs belonging to the genus Giberella, reflecting the heterogeneity of the genus Fusarium (5, 10, 11).
For diagnosis purposes, the sequencing method showed its objective value by easily and correctly identifying a nonpigmented F. solani isolate (N96144236), independent of the mycologist's experience. Also, isolates with uncertain or wrong conventional identifications were identified in agreement with the Institute of Hygiene and Epidemiology Mycology reference center identification. Finally, the sequencing method allows identification within 48 to 72 h following a 3-day culture of the isolate, as opposed to the time-consuming conventional methods. Recently, an exoantigen test was proposed for the species identification of Fusarium, which requires the production of antiserum against specific antigens (6 weeks of incubation) and the extraction of exoantigens from 10-day-old cultures (14). Our results demonstrate that 28S rDNA sequencing is a valuable method for the identification of significant Fusarium species involved in human infections.
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ACKNOWLEDGMENTS |
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We thank C. de Bièvre for providing us the Pasteur Institute collection strains and E. Gueho for confirmation of the identification of some isolates. We are indebted to F. Botterel, S. Bretagne, J. Carrière, and A. Paugam for sending us some clinical isolates.
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FOOTNOTES |
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* Corresponding author. Mailing address: Service de Parasitologie-Mycologie, CHU Amiens, 80054 Amiens Cédex 1, France. Phone: 33-3-22-45-59-75. Fax: 33-3-22-45-56-53. E-mail: chennequin{at}yahoo.com.
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REFERENCES |
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Abstract
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Materials and Methods
Results and Discussion
References
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Journal of Clinical Microbiology, November 1999, p. 3586-3589, Vol. 37, No. 11
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Copyright © 1999, American Society for Microbiology. All rights reserved.
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