MALDI-TOF mass spectrometry proteomic phenotyping of clinically relevant fungi

Abstract

Proteomics is particularly suitable for characterising human pathogens with high life cycle complexity, such as fungi. Protein content and expression levels may be affected by growth states and life cycle morphs and correlate to species and strain variation. Identification and typing of fungi by conventional methods are often difficult, time-consuming and frequently, for unusual species, inconclusive. Proteomic phenotypes from MALDI-TOF MS were employed as analytical and typing expression profiling of yeast, yeast-like species and strain variants in order to achieve a microbial proteomics population study. Spectra from 303 clinical isolates were generated and processed by standard pattern matching with a MALDI-TOF Biotyper (MT). Identifications (IDs) were compared to a reference biochemical-based system (Vitek-2) and, when discordant, MT IDs were verified with genotyping IDs, obtained by sequencing the 25-28S rRNA hypervariable D2 region. Spectra were converted into virtual gel-like formats, and hierarchical clustering analysis was performed for 274 Candida profiles to investigate species and strain typing correlation. MT provided 257/303 IDs consistent with Vitek-2 ones. However, amongst 26/303 discordant MT IDs, only 5 appeared "true". No MT identification was achieved for 20/303 isolates for incompleteness of database species variants. Candida spectra clustering agreed with identified species and topology of Candida albicans and Candida parapsilosis specific dendrograms. MT IDs show a high analytical performance and profiling heterogeneity which seems to complement or even outclass existing typing tools. This variability reflects the high biological complexity of yeasts and may be properly exploited to provide epidemiological tracing and infection dispersion patterns.

Comparison of the Microflex LT and Vitek MS Systems for Routine Identification of Bacteria by Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry

“…The ability of MALDI-TOF MS to identify yeasts and fungi has been demonstrated many times (1,14,23,26,29). In a large comparison, Bader et al found no significant difference between the Saramis and Biotyper databases when identifying yeasts (2).…”

Echinocandin Resistance in Two Candida haemulonii Isolates from Pediatric Patients

“…The isolates were identified using matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry. Samples were analyzed in triplicate, using the Putignani method (12). Scores were satisfactory (above 2) and identification was compatible with C. haemulonii for all 3 samples.…”

Microbial Tracking of Multidrug-Resistant Klebsiella Pneumoniae Isolates in a Pediatric Hospital Setting

“…The dry pellets were mixed thoroughly with 70% formic acid (50 Ill) and acetonitrile (50 Ill) (Sigma-Aldrich S.r.l. Milan, Italy) and 1.5 IIIof this suspension was air-dried, overlaid with 1.5 III of a-cyano-4-hydroxy-cinnamic acid matrix «Broker Daltonics GmbH, Bremen, Germany) and subjected to MALDI-TOF MS proteome phenotyping with a Microflex LT mass spectrometer (Broker Daltonics) using Flex Control software (version 3.0, Broker Daltonic) as described by Putignani et al (15). In particular, for each spectrum, 300 shots, in 50-shot steps from different positions of the target spot (automatic mode) were collected and analysed.…”