Cryptic Species and Azole Resistance in the Aspergillus niger Complex

Abstract

Aspergillus niger is a common clinical isolate. Multiple species comprise the Aspergillus section Nigri and are separable using sequence data. The antifungal susceptibility of these cryptic species is not known. We determined the azole MICs of 50 black aspergilli, 45 from clinical specimens, using modified EUCAST (mEUCAST) and Etest methods. Phylogenetic trees were prepared using the internal transcribed spacer, beta-tubulin, and calmodulin sequences to identify strains to species level and the results were compared with those obtained with cyp51A sequences. We attempted to correlate cyp51A mutations with azole resistance. Etest MICs were significantly different from mEUCAST MICs (P < 0.001), with geometric means of 0.77 and 2.79 mg/liter, respectively. Twenty-six of 50 (52%) isolates were itraconazole resistant by mEUCAST (MICs > 8 mg/liter), with limited cross-resistance to other azoles. Using combined beta-tubulin/calmodulin sequences, the 45 clinical isolates grouped into 5 clades, A. awamori (55.6%), A. tubingensis (17.8%), A. niger (13.3%), A. acidus (6.7%), and an unknown group (6.7%), none of which were morphologically distinguishable. Itraconazole resistance was found in 36% of the isolates in the A. awamori group, 90% of the A. tubingensis group, 33% of the A. niger group, 100% of the A. acidus group, and 67% of the unknown group. These data suggest that cyp51A mutations in section Nigri may not play as important a role in azole resistance as in A. fumigatus, although some mutations (G427S, K97T) warrant further study. Numerous cryptic species are found in clinical isolates of the Aspergillus section Nigri and are best reported as "A. niger complex" by clinical laboratories. Itraconazole resistance was common in this data set, but azole cross-resistance was unusual. The mechanism of resistance remains obscure.

Identification and Susceptibility of Aspergillus Section Nigri in China: Prevalence of Species and Paradoxical Growth in Response to Echinocandins

“…In recent years, sequence-based molecular methods have been used successfully for species identification in this group, particularly methods using the calmodulin gene, which can distinguish all species within section Nigri (4-6). Molecular studies indicated that several species in addition to A. niger were able to cause human infections; different regions had different species of pathogenic black Aspergillus, and A. niger and Aspergillus tubingensis were the most frequently identified pathogens in previous studies (2,(7)(8)(9)(10)(11)(12)(13)(14). Additionally, it is worth noting in a clinical context that azoles exhibited different activity against the two species and that isolates from different geographical regions exhibited remarkable differences in susceptibility to azoles and amphotericin B (1-13, 15).…”

Black Aspergillus species isolated from clinical and environmental samples in Iran

“…Amongst the species of the section Nigri, Aspergillus niger has been reported as the most frequent aetiological agent of otomycosis (Kaya & Kiraz, 2007). Nevertheless, the clinical implications of other species are rarely reported and they are generally misidentified as A. niger (Howard et al, 2011). Identification of clinical isolates of Aspergillus to the species level is important because different species may have dissimilar susceptibilities to antifungal drugs.…”

Determination of Azole Antifungal Drug Resistance Mechanisms Involving Cyp51a Gene in Clinical Isolates of Aspergillus fumigatus and Aspergillus niger

“…The L98H, M220 and Tandem Repeat were not detected at all in Aspergillus niger isolates and the resistance in A. niger may be due to other factors [6]. Meanwhile the resistance isolates which do not show any gene expression or L98H, M220 and TR mutation could be due to presence of other mutations such as F46Y, G89G, M172V, N248 T, D255E, L358L, E427K, C454C, L358L or efflux pump mechanism [14].…”

“…However it was not possible to amplify the other isolates with the primer pair employed. This is because A. niger is a species complex consisting of numerous different strains [14] and several other primer pairs would have to be used in order to amplify the other isolates [15][16][17][18][19][20][21][22][23][24][25][26][27][28][29].…”