Background: Aspergillus section Nigri includes several species widely distributed in the environment,
and some of them are able to cause different types of human infections. However, they are difficult to
identify based solely on morphological and culture characteristics.
The aim of the study was to perform molecular identification of Aspergillus niger clinical isolates by
sequencing the beta–tubulin gen, to build our own reference protein spectra library after MALDI-TOF
MS analysis, and to assess the validity of the library for its clinical use.
Fungal strains: From March 2014 to October 2014, 22 clinical isolates that were identified as A. niger
by morphological and culture characteristics, were collected at the Microbiology Service of the Hospital
Virgen del Rocío.
Molecular identification by Sequencing: Genomic DNA was extracted from the strains grown on
Sabouraud chloramphenicol agar plates after 48 hours of incubation at 30°C, according to the
instructions provided with the QIAamp® DNA Mini Kit (Qiagen, Courtaboeuf, France). PCR
amplification and sequencing of the partial beta-tubulin (BT) was performed using BT1 and BT4
primers. A BLAST search analysis for species identification at the NCBI genomic database
(http://blast.ncbi.nom.nih.gov/) was done.
Molecular identification by MALDI-TOF MS: The strains were grown on Sabouraud chloramphenicol
agar plates and incubated 48 hours at 30°C.
The library of reference spectra was constructed with 9 strains, using the ethanol/ formic acid
extraction procedure. For each database entry, 24 individually measured mass spectra were imported
into the software and calculated a main spectrum (MSP) that were added to the original BioTyper
Thirteen strains (previously identified by sequencing) were cultured and extracted. Three spots of each
extracted sample were identified using the 9-reference spectra library.
All the 22 isolates were identified as A. niger by their macroscopic and microscopic morphology, while
12 isolates were identified as A. niger and 10 as A. tubingensis by sequencing.
We have included 9 MSPs in own new library: 5 new entries of A. niger and 4 of A. tubingensis. Using
this new library all the 13 strains (3 spots per strain) tested, led to correct identification at the species
level with high log score values: 2,467 ± 0,138 (range from 2,116 to 2,685).
The clinical isolates including 12 (54,5%) A. niger and 10 (45,5%) A. tubingensis: were cultured from
ear and respiratory samples, suggesting that both species exhibit similar incidence rates among
All the isolates, were correctly identified by MALDI-TOF MS using own new library, so MALDI-TOF MS
allows the precise identification of A. niger and A. tubingensis in clinical settings: we observed a
significant increase from 53,84% to 100% of correct identification.
Full conference title:
- ECCMID 26th (2016)