+ Models
MYCMED-592; No. of Pages 8
Journal de Mycologie Médicale (2016) xxx, xxx—xxx
Available online at ScienceDirect
www.sciencedirect.com
ORIGINAL ARTICLE/ARTICLE ORIGINAL
Aspergillus tubingensis and Aspergillus niger
as the dominant black Aspergillus, use of
simple PCR-RFLP for preliminary
differentiation
Aspergillus tubingensis et Aspergillus niger comme Aspergillus noirs
dominants, l’utilisation d’une simple PCR-RFLP pour la
diffe´renciation pre´liminaire
a, b b c
H. Mirhendi *, F. Zarei , M. Motamedi , S. Nouripour-Sisakht
a
Department of Medical Parasitology and Mycology, School of Medicine, Isfahan University of Medical
Sciences, Isfahan, Iran
b
Department of Medical Parasitology and Mycology, School of Public Health, National Institute of Health
Research, Tehran University of Medical Sciences, Tehran, Iran
c
Cellular and Molecular Research Center, Yasuj University of Medical Sciences, Yasuj, Iran
Received 3 July 2015; received in revised form 1st December 2015; accepted 7 December 2015
KEYWORDS Summary This work aimed to identify the species distribution of common clinical and
Black Aspergillus; environmental isolates of black Aspergilli based on simple restriction fragment length polymor-
Aspergillus niger; phism (RFLP) analysis of the b-tubulin gene. A total of 149 clinical and environmental strains of
Aspergillus tubingensis; black Aspergilli were collected and subjected to preliminary morphological examination. Total
b-tubulin; genomic DNAs were extracted, and PCR was performed to amplify part of the b-tubulin gene. At
PCR-RFLP; first, 52 randomly selected samples were species-delineated by sequence analysis. In order to
Iran distinguish the most common species, PCR amplicons of 117 black Aspergillus strains were
identified by simple PCR-RFLP analysis using the enzyme TasI. Among 52 sequenced isolates, 28
were Aspergillus tubingensis, 21 Aspergillus niger, and the three remaining isolates included
Aspergillus uvarum, Aspergillus awamori, and Aspergillus acidus. All 100 environmental and
17 BAL samples subjected to TasI-RFLP analysis of the b-tubulin gene, fell into two groups,
consisting of about 59% (n = 69) A. tubingensis and 41% (n = 48) A. niger. Therefore, the method
successfully and rapidly distinguished A. tubingensis and A. niger as the most common species
* Corresponding author.
E-mail address: [email protected] (H. Mirhendi).
http://dx.doi.org/10.1016/j.mycmed.2015.12.004
1156-5233/# 2016 Elsevier Masson SAS. All rights reserved.
Please cite this article in press as: Mirhendi H, et al. Aspergillus tubingensis and Aspergillus niger as the dominant black Aspergillus, use of
simple PCR-RFLP for preliminary differentiation. Journal De Mycologie Médicale (2016), http://dx.doi.org/10.1016/j.myc- med.2015.12.004
+ Models
MYCMED-592; No. of Pages 8
2 H. Mirhendi et al.
among the clinical and environmental isolates. Although tardy, the Ehrlich test was also able to
differentiate A. tubingensis and A. niger according to the yellow color reaction specific to
A. niger. A. tubingensis and A. niger are the most common black Aspergillus in both clinical and
environmental isolates in Iran. PCR-RFLP using TasI digestion of b-tubulin DNA enables rapid
screening for these common species.
# 2016 Elsevier Masson SAS. All rights reserved.
Re´sume´ Ce travail avait pour but d’identifier la répartition des espèces des isolats cliniques et
MOTS CLÉS environnementaux communs d’Aspergillus noirs en se basant sur le simple polymorphisme de
longueur des fragments de restriction (RFLP) du gène de la -tubuline. Un total de 149 souches
Aspergillus noir ; b
cliniques et environnementales d’Aspergillus noirs ont été prélevées et soumises à l’examen
Aspergillus niger ;
morphologique préliminaire. L’ADN génomique total a été extrait et une PCR a été réalisée pour
Aspergillus tubingensis ;
amplifier une partie du gène de la -tubuline. Dans un premier temps, 52 échantillons choisis au
b-tubuline ; b
hasard ont été identifiés en espèces par analyse de séquence. Afin de distinguer les espèces les
PCR-RFLP ;
Iran plus communes, des amplicons de 117 souches d’Aspergillus noirs ont été identifiés par simple
analyse PCR-RFLP en utilisant l’enzyme TasI. Parmi 52 isolats séquencés, 28 étaient Aspergillus
tubingensis, 21 Aspergillus niger, et les trois isolats restants étaient : Aspergillus uvarum,
Aspergillus awamori, et Aspergillus acidus. Tous les 100 échantillons de l’environnement et les
17 LBA soumis à l’analyse TasI-RFLP du gène de la b-tubuline se sont retrouvés en deux groupes,
composés pour 59 % (n = 69) de A. tubingensis et 41 % (n = 48) de A. niger. Ainsi la méthode rapide
distingue avec succès A. tubingensis et A. niger, espèces les plus communes chez les isolats
cliniques et environnementaux. Bien que plus ancien, le test d’Ehrlich a également été en
mesure de différencier A. tubingensis et A. niger selon la réaction de couleur jaune spécifique à
A. niger. A. tubingensis et A. niger sont les Aspergillus noirs les plus communs dans les isolats
cliniques et environnementaux en Iran. Une PCR-RFLP utilisant TasI avec digestion de l’ADN de la
b-tubuline permet un dépistage rapide pour ces espèces communes.
# 2016 Elsevier Masson SAS. Tous droits réservés.
Introduction piperis, Aspergillus sclerotiicarbonarius, Aspergillus sclero-
tioniger, Aspergillus tubingensis and Aspergillus vadensis)
being only recently described [26].
Aspergillus species are main members of environmental
Members of Aspergillus section Nigri are reported to be
saprophytes and are typically included in fungal communities
the third most common Aspergillus species associated with
of both indoor and outdoor environments. They are normal
invasive disease and aspergilloma [4,8,22]. Aspergillus niger
components of organic debris, but can be life-threatening
has also been reported as the most frequent etiological agent
opportunistic agents in debilitated or immunocompromised
of otomycosis [15]; other species are rarely reported and
patients [3]. The genus Aspergillus includes several groups,
may be miss-identified as A. niger [34]. Since different
including Aspergillus section Nigri with several species [16],
species may have dissimilar susceptibilities to antifungal
some of which have been implicated in human disease [1].
drugs, species identification informs the choice of antifungal
The taxonomy of Aspergillus section Nigri (known as black
therapy [17,2,13]. In addition to their clinical significance,
Aspergilli) remains somewhat ill-defined. It comprises a
several black Aspergilli have agricultural importance, being
closely related group of organisms that are difficult to dis-
food spoilage organisms [21]. Ochratoxin A, produced by
tinguish based on morphological characteristics such as
some Aspergillus species in the section Nigri, is a potent
colony morphology, conidial size, and ornamentation [29].
nephrotoxin and potential carcinogen, and concern has been
Several approaches including morphological and physiologi-
raised regarding the incorporation of this compound into the
cal methods have been employed for studding this section
human and animal food chain [24].
[29]. Development of molecular DNA-based techniques such
We have already used the sequence analysis of b-tubulin
as PCR-RFLP [7], RAPD-PCR [27], and nucleotide sequencing
genes for species delineation of black Aspergilli isolates [37].
[23] for the identification of fungal strains has resulted in
In the present study, the most common species of black
reclassification of black Aspergilli, and so these tools are
Aspergilli, i.e. A. tubingensis and A. niger, isolated from
now being acknowledged as the gold standard [28]. About
clinical and environmental samples, are differentiated by
26 species have been recognized within this section, with
the use of simple PCR-RFLP analysis.
some of them (Aspergillus aculeatinus, Aspergillus aculea-
tus, Aspergillus japonicus, Aspergillus uvarum, Aspergillus
brasiliensis, Aspergillus carbonarius, Aspergillus costari- Materials and methods
caensis, Aspergillus ellipticus, Aspergillus foetidus, Asper-
gillus heteromorphus, Aspergillus homomorphus, Strains. A total of 149 clinical and environmental isolates of
Aspergillus ibericus, Aspergillus lacticoffeatus, Aspergillus black Aspergillus were used in this study. Forty-nine strains
Please cite this article in press as: Mirhendi H, et al. Aspergillus tubingensis and Aspergillus niger as the dominant black Aspergillus, use of
simple PCR-RFLP for preliminary differentiation. Journal De Mycologie Médicale (2016), http://dx.doi.org/10.1016/j.myc- med.2015.12.004
+ Models
MYCMED-592; No. of Pages 8
PCR-RFLP to differentiate A. tubingensis and A. niger 3
were isolated from patients with suspected fungal infections the amplicons was electrophoresed using a 1.5% agarose gel
referred to diagnostic laboratories in Tehran, Iran, and in TBE buffer (90 mM Tris, 90 mM boric acid, 2 mM EDTA,
additional 100 strains were recovered from soil or air in pH 8.3) and visualized under UV irradiation after ethidium
different climatic areas of the country or from some food bromide staining. Subsequently, PCR products from 52 sam-
products. Air sampling was performed in hospitals and public ples comprising 32 clinical and 20 environmental Aspergillus
places using the settled plate method on Dichloran Glycerol strains were purified and sequenced followed by species
(DG-18) Agar. Strains were preliminarily identified as A. niger identifications by BLAST analysis (http://blast.ncbi.nlm.
based on their macro-/microscopic colony appearance. nih.gov/Blast.cgi).
Total genomic DNA was isolated from each colony using Restriction patterns of b-tubulin sequences of the black
glass bead disruption [36]. Briefly, 5—10 mm of fresh colonies Aspergillus species were predicted for all known restriction
was transferred to a 1.5-mL tube with 300 mg of glass beads enzymes, using the BioEdit software version 7.2 (http://
(0.5 mm in diameter), 300 mg of lysis buffer (100 mM Tris- bioedit.software.informer.com/7.2). Predicted restriction
HCl, pH 8; 10 mM EDTA; 100 mM NaCl; 1% sodium dodecyl fragments were compared with each other in order to select
sulfate (SDS); 2% triton X-100) and 300 mL of phenol-chloro- those with the best discriminatory power. RFLP tests were
form (1:1). Samples were vortexed vigorously for 2 min, performed for a total of 117 randomly selected environmen-
centrifuged for 5 min at 5000 rpm, and the supernatant tal and clinical isolates, including 27 random samples which
was transferred to a fresh tube in which DNA was extracted had already been sequenced. Digestion was performed by
with chloroform. An identical volume of isopropanol and a incubating a 5-mL aliquot of each PCR product with 5 U
0.1-volume of 3M sodium acetate (pH 5.2) were added to the enzyme in a final reaction volume of 15 mL at 65 8C for
supernatant, and after incubation at À20 8C for 30 min, the 2 h, and digested DNA was analyzed by electrophoresis using
mixture was centrifuged for 15 min at 12,000 rpm. The pre- a 2% agarose gel.
cipitant was washed with cold 70% ethanol, dried in air, The Ehrlich test (detection of fungal alkaloids reacting with
dissolved in 50 mL of water, and stored at À20 8C until use. Ehrlich reagent) was used by applying the filter paper method
The b-tubulin gene was amplified using Bt2a and Bt2b [11]. A 4-cm piece of Whatman filter paper wetted with Ehrlich
primers [12] as described previously [37]. A 5-mL aliquot of reagent (2 g of 4-dimethylamino-benzaldehyde in 85 mL
Figure 1 Microscopy of examples of black Aspergillus species examined in this study. Photos A to D represent A. niger,
A. tubingensis, A. acidus, and A. uvarum, respectively.
Exemples microscopiques d’espe`ces d’Aspergillus noirs´etudie´s dans ce travail. Les photos A`a D repre´sentent A. niger, A. tubingensis,
A. acidus, et A. uvarum, respectivement.
Please cite this article in press as: Mirhendi H, et al. Aspergillus tubingensis and Aspergillus niger as the dominant black Aspergillus, use of
simple PCR-RFLP for preliminary differentiation. Journal De Mycologie Médicale (2016), http://dx.doi.org/10.1016/j.myc- med.2015.12.004
+ Models
MYCMED-592; No. of Pages 8
4 H. Mirhendi et al.
ethanol added to 15 mL 10 N HCl) was placed on the mycelial A. niger, A. awamorii, and A. acidus by b-tubulin sequencing,
side of a 1 Â 1 cm agar plug cut from colonies grown on Czapek produced biseriate phialides, while A. uvarum exhibited
Yeast Extract agar (CYA) for one week [28]. A yellow ring uniseriate phialides. The ornamentation of the conidia was
appeared after about 10 min in isolates of A. niger, while no also characteristic for some species such as A. tubingensis
color change was observed for isolates of A. tubingensis. and A. niger, which produced conidia with a spiny appea-
rance, while A. awamori, A. acidus, and A. uvarum were
characterized by smooth conidia. Fig. 2 shows the colonies of
Results
some isolated black Aspergillus on Sabouraud dextrose agar
(SDA) after 5—7 days of incubation at 25 8C. The different
In this study, a total of 49 clinical and 100 environmental species exhibited slightly different growth characteristics.
strains morphologically recognized as black Aspergillus were Strains identified as A. niger and A. tubingensis had shared
subjected to molecular identification. In cultures, the strains colony characters in SDA and CYA that was not helpful to
presented microscopic characteristics such as dark-brown to distinguish them from each other.
black conidia, spherical vesicles, and hyaline or lightly pig- Using the universal fungal b-tubulin primer pair, a 500—
mented hyphae near the apex. These morphological features 550 base pair (bp) fragment was successfully amplified in all
were generally shared among most strains (Fig. 1). Most tested isolates, while no PCR-amplification was seen in nega-
isolates, which were later identified as A. tubingensis, tive controls. Fig. 3 (A) shows agarose gel electrophoresis of
Figure 2 Examples of colonies of Aspergillus section Nigri isolated on SDA after 5 days of incubation. A, B, C, and D represent
A. niger; E, F, G, and H represent A. tubingensis, and I represents A. acidus.
Exemples de colonies d’Aspergillus section Nigri isole´es sur SDA apre`s 5 jours d’incubation. A, B, C et D repre´sentent A. niger ; E, F, G
et H repre´sentent A. tubingensis et I repre´sente A. acidus.
Please cite this article in press as: Mirhendi H, et al. Aspergillus tubingensis and Aspergillus niger as the dominant black Aspergillus, use of
simple PCR-RFLP for preliminary differentiation. Journal De Mycologie Médicale (2016), http://dx.doi.org/10.1016/j.myc- med.2015.12.004
+ Models
MYCMED-592; No. of Pages 8
PCR-RFLP to differentiate A. tubingensis and A. niger 5
Figure 3 Agarose gel electrophoresis of b-tubulin PCR products. A. Before digestion with TasI: lanes 1—7 are example samples.
Lanes 8 and 9 are negative controls, and lane M is a 100-bp molecular size marker. B. After digestion with TasI: lanes 1, 2, 4, and 5 are
A. niger, lane 3 is A. tubingensis, and lane M is a 100-bp molecular size marker.
´Electrophore`se en gel d’agarose des produits de la b-tubuline. A. Avant digestion avec TasI : les bandes 1 `a 7 sont des exemples
d’e´chantillons. Bandes 8 et 9 sont des controˆles ne´gatifs et la bande M est un marqueur de taille mole´culaire de 100-pb. B. Apre`s
digestion par TasI : bandes 1, 2, 4 et 5 sont A. niger, bande 3 : A. tubingensis et bande M est un marqueur mole´culaire de taille de
100-pb.
PCR products from isolated black Aspergillus species. The After analysis of nearly all commercially available res-
BLAST analysis of the sequences indicated that 28 (53.8%) triction enzymes, TasI was selected as one of the appropriate
and 21 (40.3%) isolates were A. tubingensis and A. niger, enzymes for differentiation between the two dominant spe-
respectively. Accounting for about 6% of all sequenced sam- cies of black Aspergillus isolated in this study, A. niger and
ples, three other sequences represented A. uvarum, A. tubingensis. Fragment sizes of PCR products of all species
A. awamori, and A. acidus (Table 1). The sequences were identified by sequencing in this study, before and after
deposited in GenBank and assigned as the accession digestion with TasI, are shown in Table 2. A total of
numbers KT965680 to KT965724. 117 PCR products subjected to PCR-RFLP including all
Table 1 Summary of the results on species identification obtained for tested samples in this study.
Re´sume´ des re´sultats de l’identification des espe`ces des ´echantillons de cette ´etude.
Source Identified by sequencing Identified by PCR-RFLP
Number of Identified species Number of Identified species
tested isolates tested isolates
Clinical samples (49)
Nail (19) 19 A. tubingensis (9) 0 —
A. niger (8)
A. uvarum (1)
A. awamori (1)
BAL, sputum, palate and nose (24) 7 A. tubingensis (7) 17 A. tubingensis (8)
A. niger (9)
Cerumen (4) 4 A. tubingensis (2) 0 0
A. niger (2)
Skin lesions (2) 2 A. niger (2) 0 0
Environmental samples (100)
Air (36) 10 A. tubingensis (6) 36 A. tubingensis (24)
A. niger (4) A. niger (12)
Spice (22) 6 A. tubingensis (3) 22 A. tubingensis (10)
A. niger (3) A. niger (12)
Grape (7) 2 A. tubingensis (1) 7 A. tubingensis (2)
A. niger (1) A. niger (5)
Soil (29) 2 A. niger (1) 29 A. tubingensis (22)
A. acidus (1) A. niger (7)
Dried fruit (4) 0 0 4 A. tubingensis (2)
A. niger (2)
Grain (2) 0 0 2 A. tubingensis (1)
A. niger (1)
Total number
149 52 52 117 117
Please cite this article in press as: Mirhendi H, et al. Aspergillus tubingensis and Aspergillus niger as the dominant black Aspergillus, use of
simple PCR-RFLP for preliminary differentiation. Journal De Mycologie Médicale (2016), http://dx.doi.org/10.1016/j.myc- med.2015.12.004
+ Models
MYCMED-592; No. of Pages 8
6 H. Mirhendi et al.
Table 2 In silico TasI-RFLP analysis of b-tubulin for black Aspergillus species isolated in this study.
In silico TasI-RFLP analyse de la b-tubuline pour les Aspergillus noirs isole´s dans cette ´etude.
Species Example of GenBank PCR product size in bp PCR product size in bp
accession number before digestion with TasI after digestion with TasI
A. niger JX4633191 555 78, 141, 336
A. tubingensis KF434100 555 219, 336
A. acidus KC4336731 558 221, 337
A. uvarum JQ3179721 540 63, 118, 359
A. awamori HQ2855991 557 78, 141, 338
100 environmental and 17 BAL samples fell into two groups, In the present study, the black Aspergilli isolated from
consisting of 59% (n = 69) A. tubingensis and 41% (n = 48) clinical and environment samples in Iran were identified using
A. niger. A total of 27 isolates randomly selected from those a combination of different methods with a view to develop a
already identified by b-tubulin sequencing were subjected to better understanding of the distribution of species profiles.
PCR-TasI-RFLP by which 26 of 27 samples had identical Since b-tubulin is acknowledged as a valid marker for species
results. Only one isolate identified as A. acidus by sequenc- differentiation of black Aspergilli, we carried out sequence
ing, was identified as A. tubingensis by TasI-RFLP analysis analysis, targeting molecular identification of 52 isolates,
(Table 1). An example of agarose gel electrophoresis of PCR- among which A. tubingensis and A. niger were, by far, the
RFLP products of representative isolates of Aspergillus is most common species of black Aspergillus. The details of this
shown in Fig. 3 (B). As seen, the bands generated corres- sequence analysis have already been reported [37].
ponded exactly to the predicted sizes (Table 2). Therefore, we selected the enzyme for RFLP analysis,
The Ehrlich test was performed on examples of primarily with a view to discriminating between these two
A. tubingensis and A. niger isolates since these were the species. Our results confirm that these species display dif-
most abundant species in our study. A clear difference in ferent RFLP-based profiles (Fig. 3B). PCR products from
alkaloid production was observed between the species. The A. niger were cleaved into three fragments of 78, 141 and
test yielded a yellow reaction (positive) for A. niger and no 336 bp by TasI; meanwhile, there was only one restriction
color (negative) for A. tubingensis (Fig. 4). site for this enzyme in the sequence of A. tubingensis, for
which only two fragments were produced (219 and 336 bp).
Discussion Also, A. uvarum was cleaved into three fragments of 63, 118,
359 bp. The enzyme digestion pattern was the same for
A. tubingensis/A. acidus and A. niger/A. awamori. Given
Studies have suggested that a significant proportion of cli-
the fact that there was only one isolate of A. awamori and
nical isolates considered as A. niger are indeed other mem-
A. acidus among the 52 isolates sequenced, it appears that b-
bers of black Aspergilli, such as A. tubingensis,
tubulin-PCR followed by TasI-RFLP generally successfully
A. brasiliensis, and A. foetidus [2]. Some species have
differentiates and identifies isolates as A. niger or
distinct biochemical properties, such as those pertaining
A. tubingensis, being an easy and inexpensive tool for pre-
to nutritional growth conditions and hydrolase differences
liminary differentiation of black Aspergillus.
[20]. Production of secondary metabolites is often unique for
Recently, clinical isolates thought to belong to A. niger
species within Aspergillus section Nigri and could be used for
were re-classified by genetic tools as A. tubingensis,
identification; however, it is not yet possible to differentiate
A. awamori, A. uvarum, and A. brasiliensis [13,19,25,31].
the species solely on metabolic properties. Meanwhile, the
According to our results, A. niger is no longer the dominant
development of molecular diagnostic tools has facilitated
black Aspergillus; instead, A. tubingensis comprises more
correct species determination of black Aspergilli [3].
than half of the strains that have usually been assigned to
A. niger. Likewise, Howard et al. examined 43 black Asper-
gilli derived from various clinical sources by sequence ana-
lysis of the internal transcribed spacer (ITS) region of the
ribosomal RNA gene, partial calmodulin, and b-tubulin
sequences and found that 69.7% of the isolates belonged
to A. niger and A. awamori, 18.6% to A. tubingensis, 7.0% to
A. foetidus, and 4.7% to an undescribed species [14].
Otomycosis represents an infection, which is typically
caused by black Aspergilli in tropical and semitropical cli-
mates [10]. Vennewald et al. isolated fungal strains from the
middle ear of immunocompetent patients with chronic otitis
media, in which A. niger was observed in three out of five
cases [33]. Sequence data in this study indicated that in
Figure 4 Ehrlich color reaction. A. Positive color reaction addition to A. niger, A. tubingensis is also able to cause ear
(yellow) in A. niger. B. Negative color reaction in A. tubingensis. infections in Iran. Likewise, Szigeti et al. by using partial
Re´action colore´e d’Ehrlich. A. Re´action positive (jaune) avec analysis of the calmodulin gene sequence, suggested that in
A. niger. B. Re´action ne´gative avec A. tubingensis. addition to A. niger and A. tubingensis, A. awamori is also
Please cite this article in press as: Mirhendi H, et al. Aspergillus tubingensis and Aspergillus niger as the dominant black Aspergillus, use of
simple PCR-RFLP for preliminary differentiation. Journal De Mycologie Médicale (2016), http://dx.doi.org/10.1016/j.myc- med.2015.12.004
+ Models
MYCMED-592; No. of Pages 8
PCR-RFLP to differentiate A. tubingensis and A. niger 7
capable of causing otomycosis in Iran [32] and Hungary [31]. References
In mycotic keratitis, although the primary causative agent
within the genus is A. flavus, black Aspergilli appear to be
[1] Abarca ML, Accensi F, Cano J, Cabanes FJ. Taxonomy and
the most frequent pathogens in certain geographic regions
significance of black Aspergilli. Antonie Van Leeuwenhoek
[5]. From Aspergillus section Nigri, only A. niger has been 2004;86:33—49.
reported to date as a possible causative agent of fungal [2] Alcazar-Fuoli L, Mellado E, Alastruey-Izquierdo A, Cuenca-
keratitis [5]. The causative agents of two reported Indian Estrella M, Rodriguez-Tudela JL. Species identification and
antifungal susceptibility patterns of species belonging to As-
cases of keratitis were identified as A. brasiliensis based on
pergillus section Nigri. Antimicrob Agents Chemother 2009;53:
partial sequence analysis of the b-tubulin gene [19], suggest-
4514—7.
ing that this newly described species may be responsible for a
[3] Barton RC. Laboratory diagnosis of invasive aspergillosis: from
significant proportion of corneal infections caused by black
diagnosis to prediction of outcome. Scientifica 2013.
Aspergilli.
[4] Bathoorn E, Salazar NE, Sepehrkhouy S, Meijer M, de Cock H,
Non-dermatophytic molds account for 1.5%—6% of ony-
Haas PJ. Involvement of the opportunistic pathogen Aspergillus
chomycosis [18] and are most frequently seen in elderly, in
tubingensis in osteomyelitis of the maxillary bone: a case
patients with skin diseases, or in immunocompromised report. BMC Infect Dis 2013;13:59.
patients. Although representatives of the section Nigri are [5] Bhaskar M, et al. Black Aspergilli in tropical infections. Rev Med
considered non-dermatophytic molds, their prevalence in Microbiol 2008;19:65—78.
[6] Bonifaz A, Cruz-Aguilar P, Ponce RM. Onychomycosis by molds.
nail infections is low according to the total number of
Report of 78 cases. Eur J Dermatol 2007;17:70—2.
culture-proven cases of onychomycosis [6]. However, their
[7] de Vries RP, et al. Aspergillus vadensis, a new species of the
proportion within non-dermatophytic onychomycosis can be
group of black Aspergilli. Antonie Van Leeuwenhoek
high [30]. Among Aspergillus species, A. niger predominated
2005;87:195—203.
in nail specimens according to a survey performed in New
[8] Denning DW. Invasive aspergillosis. Clin Infect Dis 1998;781—803.
Delhi [35]. A. niger was also found to be able to cause
[9] Eenglish MP. Nails and fungi. Br J Dermatol 1976;94:697—701.
subungual onychomycosis in Italy [5]. According to English,
[10] Fasunla J, Ibekwe T, Onakoya P. Otomycosis in western Nigeria.
non-dermatophytic mold can be considered as a pathogen of Mycoses 2008;51:67—70.
onychomycosis only when hyphae or spores are seen on [11] Frisvad JC, Samson RA. Polyphasic taxonomy of Penicillium
microscopic examination and the same strain is identified subgenus Penicillium. A guide to identification of food and air-
borne terverticillate Penicillia and their mycotoxins. Stud
through repeated cultures [9]. In our study, the results of
Mycol 2004;49:174.
sequencing of nail samples clearly indicated A. tubingensis
[12] Glass NL, Donaldson GC. Development of primer sets designed
and A. niger as predominating species. In the initial evalua-
for use with the PCR to amplify conserved genes from filamen-
tion of a nail sample, hyphae were observed by the KOH test.
tous ascomycetes. Appl Environ Microbiol 1995;61:1323—30.
Sequencing result of the grown colonies matched 100% with
[13] Hendrickx M, Beguin H, Detandt M. Genetic re-identification
A. uvarum. The details of the case are reported elsewhere
and antifungal susceptibility testing of Aspergillus section Nigri
[38]. Also, A. acidus was isolated from a soil sample, a
strains of the BCCM/IHEM collection. Mycoses 2012;55:148—55.
species that was identified as the cause of human [14] Howard S, Harrison E, Bowyer P, Denning D. Molecular identifi-
infections [2]. cation of clinical black Aspergillus isolates and azole resis-
Although micro-morphological structures can be helpful, tance. 3rd Advances Against Aspergillosis Conference 2008.
p. 16—8.
in A. niger and its related taxa, it is difficult to distinguish the
[15] Kaya AD, Kiraz N. In vitro susceptibilities of Aspergillus spp.
described species. Nevertheless, A. carbonarius and the
causing otomycosis to amphotericin B, voriconazole and itra-
uniseriate species (A. uvarum, A. aculeatus, and A. japoni-
conazole. Mycoses 2007;50:447—50.
cus) can be microscopically distinguished by vesicle and
[16] Klich MA. Health effects of Aspergillus in food and air. Toxicol
conidial size plus ornamentation [1]. Differentiation of clo-
Ind Health 2009;25:657—67.
sely related species such as A. niger and A. awamori is a
[17] Li Y, Wan Zh, Liu W, Li R. Identification and susceptibility of
challenge because of the existence of very similar morpho-
Aspergillus section Nigri in China: prevalence of species and
logical characters [5]. Fortunately, A. niger and paradoxical growth in response to Echinocandins. JCM
A. tubingensis, can be easily but tardily differentiated by 2015;53:702—5.
the Ehrlich test, in which A. niger triggers a yellow reaction, [18] Malini A, Oudeacoumar P, Udayashankar C. Onychomycosis due
to Trichosporon mucoides. Indian J Dermatol Venereol Leprol
while A. tubingensis does not.
2011;77:76.
[19] Manikandan P, et al. Keratitis caused by the recently described
Conclusion
new species Aspergillus brasiliensis: two case reports. J Med
Case Rep 2010;4:68.
A. tubingensis and A. niger are the most common black [20] Meijer M, Houbraken J, Dalhuijsen S, Samson R, de Vries R.
Growth and hydrolase profiles can be used as characteristics to
Aspergillus in both clinical and environmental isolates in
distinguish Aspergillus niger and other black Aspergilli. Stud
Iran. PCR-RFLP using TasI digestion of b-tubulin DNA enables
Mycol 2011;69:19—30.
rapid screening of these two species. Although tardy, the
[21] Noonim P, Mahakarnchanakul W, Varga J, Frisvad JC, Samson
Ehrlich test was also able to differentiate A. tubingensis and
RA. Two novel species of Aspergillus section Nigri from Thai
A. niger.
coffee beans. Int J Syst Evol Microbiol 2008;58:1727—34.
[22] Pappas PG, et al. Invasive fungal infections among organ trans-
Disclosure of interest plant recipients: results of the Transplant-Associated Infection
Surveillance Network (TRANSNET). Clin Infect Dis
2010;50:1101—11.
The authors declare that they have no competing interest.
Please cite this article in press as: Mirhendi H, et al. Aspergillus tubingensis and Aspergillus niger as the dominant black Aspergillus, use of
simple PCR-RFLP for preliminary differentiation. Journal De Mycologie Médicale (2016), http://dx.doi.org/10.1016/j.myc- med.2015.12.004
+ Models
MYCMED-592; No. of Pages 8
8 H. Mirhendi et al.
[23] Pel HJ, et al. Genome sequencing and analysis of the versatile [31] Szigeti G, Kocsube S, Doczi I, Bereczki L, Vagvolgyi C, Varga J.
cell factory Aspergillus niger CBS 513.88. Nat Biotechnol Molecular identification and antifungal susceptibilities of black
2007;25:221—31. Aspergillus isolates from otomycosis cases in Hungary. Mycopa-
[24] Perrone G, Mule G, Susca A, Battilani P, Pietri A, Logrieco A, thologia 2012;174:143—7.
et al. production and amplified fragment length polymorphism [32] Szigeti G, et al. Species assignment and antifungal susceptibili-
analysis of Aspergillus carbonarius, Aspergillus tubingensis, ties of black Aspergilli recovered from otomycosis cases in Iran.
and Aspergillus niger strains isolated from grapes in Italy. Appl Mycoses 2012;55:333—8.
Environ Microb 2006;72:680—5. [33] Vennewald I, Schonlebe J, Klemm E. Mycological and histologi-
[25] Perrone G, et al. Aspergillus uvarum sp. nov., an uniseriate cal investigations in humans with middle ear infections. Myco-
black Aspergillus species isolated from grapes in Europe. Int J ses 2003;46:12—8.
Syst Evol Microbiol 2008;58:1032—9. [34] Williams B, Popoola B, Ogundana S. A possible new pathogenic
[26] Perrone G, Stea G, Epifani F, Varga J, Frisvad JC, Samson RA. Aspergillus isolation and general mycological properties of the
Aspergillus niger contains the cryptic phylogenetic species fungus. Afr J Med Med Sci 1983;13:111—5.
A. awamori. Fungal Biol 2011;115:1138—50. [35] Xess I, Mohanty S, Jain N, Banerjee U. Prevalence of Aspergillus
[27] Samson RA, Pitt JI. Integration of modern taxonomic methods species in clinical samples isolated in an Indian tertiary care
for Penicillium and Aspergillus classification. CRC Press; 2000. hospital. Indian J Med Sci 2004;58:513.
[28] Samson RA, Noonim P, Meijer M, Houbraken J, Frisvad JC, Varga [36] Yamada Y, et al. Comparison of different methods for extrac-
J. Diagnostic tools to identify black Aspergilli. Stud Mycol tion of mitochondrial DNA from human pathogenic yeasts. Jpn J
2007;59:129—45. Infect Dis 2002;55:122—5.
[29] Silva DM, Batista LR, Rezende EF, Fungaro MHP, Sartori D, Alves [37] Zarei F, et al. Black Aspergillus species isolated from clinical
E. Identification of fungi of the genus Aspergillus section Nigri and environmental samples in Iran. J Med Microbiol 2015.
using polyphasic taxonomy. Braz J Microbiol 2011;42:761—73. http://dx.doi.org/10.1099/jmm.0.000166.
[30] Surjushe A, Kamath R, Oberai C, Saple D, Thakre M, Dharmshale [38] Zarei F, Mirhendi H, Fakhim H, Geramishoar M. The first case of
S, et al. A clinical and mycological study of onychomycosis in onychomycosis due to Aspergillus uvarum (section Nigri). My-
HIV infection. Indian J Dermatol Venereol Leprol 2007;73:397. coses 2015;58:239—42.
Please cite this article in press as: Mirhendi H, et al. Aspergillus tubingensis and Aspergillus niger as the dominant black Aspergillus, use of
simple PCR-RFLP for preliminary differentiation. Journal De Mycologie Médicale (2016), http://dx.doi.org/10.1016/j.myc- med.2015.12.004