Multilocus variable-number tandem-repeat analysis of clinical isolates of Aspergillus flavus from Iran reveals the first cases of Aspergillus minisclerotigenes associated with human infection

Abstract

Background: Aspergillus flavus is intensively studied for its role in infecting crop plants and contaminating produce with aflatoxin, but its role as a human pathogen is less well understood. In parts of the Middle East and India, A. flavus surpasses A. fumigatus as a cause of invasive aspergillosis and is a significant cause of cutaneous, sinus, nasal and nail infections.

Methods: A collection of 45 clinical and 10 environmental A. flavus isolates from Iran were analysed using Variable-Number Tandem-Repeat (VNTR) markers with MICROSAT and goeBURST to determine their genetic diversity and their relatedness to clinical and environmental A. flavus isolates from Australia. Phylogeny was assessed using partial β-tubulin and calmodulin gene sequencing, and mating type was determined by PCR. Antifungal susceptibility testing was performed on selected isolates using a reference microbroth dilution method.

Results: There was considerable diversity in the A. flavus collection, with no segregation on goeBURST networks according to source or geographic location. Three Iranian isolates, two from sinus infections and one from a paranasal infection grouped with Aspergillus minisclerotigenes, and all produced B and G aflatoxin. Phylogenic analysis using partial β-tubulin and calmodulin sequencing confirmed two of these as A. minisclerotigenes, while the third could not be differentiated from A. flavus and related species within Aspergillus section flavi. Based on epidemiological cut-off values, the A. minisclerotigens and A. flavus isolates tested were susceptible to commonly used antifungal drugs.

Conclusions: This is the first report of human infection due to A. minisclerotigenes, and it raises the possiblity that other species within Aspergillus section flavi may also cause clinical disease. Clinical isolates of A. flavus from Iran are not distinct from Australian isolates, indicating local environmental, climatic or host features, rather than fungal features, govern the high incidence of A. flavus infection in this region. The results of this study have important implications for biological control strategies that aim to reduce aflatoxin by the introduction of non-toxigenic strains, as potentially any strain of A. flavus, and closely related species like A. minisclerotigenes, might be capable of human infection.

Figure 1

Neighbor-joining phylograms of clinical and environmental Aspergillus flavus isolates based on MLV data. A) Iranian and Australian isolates; B) Inclusion of additional Group II/A. minisclerotigenes isolates. Iranian clinical isolates 66401 and 66161 lie within the Group II/A. minisclerotigenes group, while isolate 65728 lies on the outside of this group.

Figure 2

goeBURST networks of clinical and environmental Aspergillus flavus isolates. Networks were drawn using the Minimum Spanning Tree expansion implemented in PHYLOVIZ. Circles represent MLV genotypes and are proportional to the number of isolates with the same genotype; MLV genotypes are numbered as shown in Table  1. Solid lines show genotypes joined at the double locus variant (DLV) level; dashed lines show genotypes joined at the single locus variant (SLV) level. Networks are integrated with A) taxonomic; B) geographic; C) clinical; and D) toxin data. White arrows: Iranian A. minisclerotigenes isolates 66041 and 66161; black arrow: Iranian isolate 65728.

Figure 3

Phylogenetic placement of Group II/A. minisclerotigenes -like isolates in Aspergillus section flavi. A 444 bp fragment of the β-tubulin gene was amplified from Group II/A. minisclerotigenes-like isolates and aligned with published Aspergillus section flavi sequences from [10], using A. leporis as an outgroup. Maximum likelihood trees were drawn using the PAUP 4.0 plug-in for Geneious R6.1.6; bootstrap support was obtained using 1000 replicates. Isolates sequenced in this study are indicated by a dot at the terminus of their branch: white = strains from culture collections; grey = Group II isolates from our collection; black = clinical Iranian isolates. Most of the Group II isolates from our collection along with Iranian clinical isolates 66041 and 66161 form a distinct cluster with 95% bootstrap support with isolates previously characterized as A. minisclerotigenes[10], including isolate FRR5309. Iranian clinical isolate 65728 groups away from this cluster with A. flavus and closely related species. Group II-like isolates LA 2–5 SB and BN038 G SGB group within the A. flavus cluster and with A. parvisclerotigenes, respectively. Accession numbers for sequences are listed after the name of the relevant isolate. *denotes FRR.