Species Identification and Delineation of Pathogenic Mucorales by Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry

Abstract

This study aimed to validate the effectiveness of matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS)-based identification of filamentous fungi of the order Mucorales. A total of 111 isolates covering six genera preserved at the Research Center for Medical Mycology of Peking University were selected for MALDI-TOF MS analysis. We emphasized the study of 23 strains of Mucor irregularis predominantly isolated from patients in China. We first used the Bruker Filamentous Fungi library (v1.0) to identify all 111 isolates. To increase the identification rate, we created a compensatory in-house database, the Beijing Medical University (BMU) database, using 13 reference strains covering 6 species, including M. irregularis, Mucor hiemalis, Mucor racemosus, Cunninghamella bertholletiae, Cunninghamella phaeospora, and Cunninghamella echinulata All 111 isolates were then identified by MALDI-TOF MS using a combination of the Bruker library and BMU database. MALDI-TOF MS identified 55 (49.5%) and 74 (66.7%) isolates at the species and genus levels, respectively, using the Bruker Filamentous Fungi library v1.0 alone. A combination of the Bruker library and BMU database allowed MALDI-TOF MS to identify 90 (81.1%) and 111 (100%) isolates at the species and genus levels, respectively, with a significantly increased accuracy rate. MALDI-TOF MS poorly identified Mucorales when the Bruker library was used alone due to its lack of some fungal species. In contrast, this technique perfectly identified M. irregularis after main spectrum profiles (MSPs) of relevant reference strains were added to the Bruker library. With an expanded Bruker library, MALDI-TOF MS is an effective tool for the identification of pathogenic Mucorales.

Keywords: MALDI-TOF MS; Mucorales; identification.

FIG 1

Cluster analysis based on the main MSPs of all 111 isolates, which belong to six different clinically relevant genera of Mucorales. The distance levels of each subdivision are shown in arbitrary units, and the isolates of the Lichtheimia and Cunninghamella genera are shown as spp.

FIG 2

Rhizopus spp. (A) MSP dendrogram based on MALDI-TOF MS analysis. (B) ML tree obtained from ITS sequences of R. arrhizus and R. microsporus. Numbers of ML bootstrap values above 70% are shown at the nodes.

FIG 3

Mucor spp. (A) MSP dendrogram based on MALDI-TOF MS analysis. (B) ML tree based on ITS sequences of M. circinelloides, M. irregularis, M. hiemalis, and M. racemosus. Numbers of ML bootstrap values above 70% are shown at the nodes.