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