All organisms usually isolated in our laboratory are now routinely identified by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) using the Andromas software. The aim of this study was to describe the use of this strategy in a routine clinical microbiology laboratory. The microorganisms identified included bacteria, mycobacteria, yeasts and Aspergillus spp. isolated on solid media or extracted directly from blood cultures. MALDI-TOF MS was performed on 2665 bacteria isolated on solid media, corresponding to all bacteria isolated during this period except Escherichia coli grown on chromogenic media. All acquisitions were performed without extraction. After a single acquisition, 93.1% of bacteria grown on solid media were correctly identified. When the first acquisition was not contributory, a second acquisition was performed either the same day or the next day. After two acquisitions, the rate of bacteria identified increased to 99.2%. The failures reported on 21 strains were due to an unknown profile attributed to new species (9) or an insufficient quality of the spectrum (12). MALDI-TOF MS has been applied to 162 positive blood cultures. The identification rate was 91.4%. All mycobacteria isolated during this period (22) were correctly identified by MALDI-TOF MS without any extraction. For 96.3% and 92.2% of yeasts and Aspergillus spp., respectively, the identification was obtained with a single acquisition. After a second acquisition, the overall identification rate was 98.8% for yeasts (160/162) and 98.4% (63/64) for Aspergillus spp. In conclusion, the MALDI-TOF MS strategy used in this work allows a rapid and efficient identification of all microorganisms isolated routinely.

Whole-cell matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) is a rapid method for identification of microorganisms that is increasingly used in microbiology laboratories. This identification is based on the comparison of the tested isolate mass spectrum with reference databases. Using Neisseria meningitidis as a model organism, we showed that in one of the available databases, the Andromas database, 10 of the 13 species-specific biomarkers correspond to ribosomal proteins. Remarkably, one biomarker, ribosomal protein L32, was subject to inter-strain variability. The analysis of the ribosomal protein patterns of 100 isolates for which whole genome sequences were available, confirmed the presence of inter-strain variability in the molecular weight of 29 ribosomal proteins, thus establishing a correlation between the sequence type (ST) and/or clonal complex (CC) of each strain and its ribosomal protein pattern. Since the molecular weight of three of the variable ribosomal proteins (L30, L31 and L32) was included in the spectral window observed by MALDI-TOF MS in clinical microbiology, i.e., 3640-12000 m/z, we were able by analyzing the molecular weight of these three ribosomal proteins to classify each strain in one of six subgroups, each of these subgroups corresponding to specific STs and/or CCs. Their detection by MALDI-TOF allows therefore a quick typing of N. meningitidis isolates.

Matrix-associated laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) is a rapid and simple microbial identification method. Previous reports using the Biotyper system suggested that this technique requires a preliminary extraction step to identify Gram-positive rods (GPRs), a technical issue that may limit the routine use of this technique to identify pathogenic GPRs in the clinical setting. We tested the accuracy of the MALDI-TOF MS Andromas strategy to identify a set of 659 GPR isolates representing 16 bacterial genera and 72 species by the direct colony method. This bacterial collection included 40 C. diphtheriae , 13 C. pseudotuberculosis , 19 C. ulcerans , and 270 other Corynebacterium isolates, 32 L. monocytogenes and 24 other Listeria isolates, 46 Nocardia , 75 Actinomyces , 18 Actinobaculum , 11 Propionibacterium acnes , 18 Propionibacterium avidum , 30 Lactobacillus , 21 Bacillus , 2 Rhodococcus equi , 2 Erysipelothrix rhusiopathiae , and 38 other GPR isolates, all identified by reference techniques. Totals of 98.5% and 1.2% of non- Listeria GPR isolates were identified to the species or genus level, respectively. Except for L. grayi isolates that were identified to the species level, all other Listeria isolates were identified to the genus level because of highly similar spectra. These data demonstrate that rapid identification of pathogenic GPRs can be obtained without an extraction step by MALDI-TOF mass spectrometry.

Species of the Pseudallescheria boydii/Scedosporium apiospermum complex (PSC) are emerging fungal pathogens able to chronically colonize the airways of patients with cystic fibrosis (CF). As P. boydii was found more frequently colonizing the lungs of CF patients in France than in other European countries in a previous report, the present study was conducted in order to clarify distribution of PSC species in France and to characterize their natural habitat. The highest densities of PSC isolates were found in human-impacted areas, i.e. agricultural areas, fluids obtained from wastewater treatment plants, playgrounds and industrial areas. PSC was not detected from soil samples collected in forests. Most PSC culture-positive soil samples exhibited a pH range of 6-8. Scedosporium dehoogii, the most abundant species, was detected in all human-impacted area types except vineyards, whereas Scedosporium aurantiacum was mostly found in agricultural areas. Pseudallescheria boydii and S. apiospermum were predominantly isolated from seashores and playgrounds respectively. Pseudallescheria minutispora was found only once from a playground. This study highlights potential sources of contamination of the patients, especially in the CF context.

An increasing number of infections due to Pseudallescheria/Scedosporium species has been reported during the past decades, both in immunocompromised and immunocompetent patients. Additionally, these fungi are now recognized worldwide as common agents of fungal colonization of the airways in cystic fibrosis patients, which represents a risk factor for disseminated infections after lung transplantation. Currently six species are described within the Pseudallescheria/Scedosporium genus, including Scedosporium prolificans and species of the Pseudallescheria/Scedosporium apiospermum complex (i.e. S. apiospermum sensu stricto, Pseudallescheria boydii, Scedosporium aurantiacum, Pseudallescheria minutispora and Scedosporium dehoogii). Precise identification of clinical isolates at the species level is required because these species differ in their antifungal drug susceptibility patterns. Matrix-assisted laser desorption ionization (MALDI)-time of flight (TOF)/mass spectrometry (MS) is a powerful tool to rapidly identify moulds at the species level. We investigated the potential of this technology to discriminate Pseudallescheria/Scedosporium species. Forty-seven reference strains were used to build a reference database library. Profiles from 3-, 5- and 7-day-old cultures of each reference strain were analysed to identify species-specific discriminating profiles. The database was tested for accuracy using a set of 64 clinical or environmental isolates previously identified by multilocus sequencing. All isolates were unequivocally identified at the species level by MALDI-TOF/MS. Our results, obtained using a simple protocol, without prior protein extraction or standardization of the culture, demonstrate that MALDI-TOF/MS is a powerful tool for rapid identification of Pseudallescheria/Scedosporium species that cannot be currently identified by morphological examination in the clinical setting.

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