Identification of filamentous fungi isolates by MALDI-TOF mass spectrometry: clinical evaluation of an extended reference spectra library.

The identification of filamentous fungi by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) relies mainly on a robust and extensive database of reference spectra. To this end, a large in-house library containing 760 strains and representing 472 species was built and evaluated on 390 clinical isolates by comparing MALDI-TOF MS with the classical identification method based on morphological observations. The use of MALDI-TOF MS resulted in the correct identification of 95.4% of the isolates at species level, without considering LogScore values. Taking into account the Brukers' cutoff value for reliability (LogScore >1.70), 85.6% of the isolates were correctly identified. For a number of isolates, microscopic identification was limited to the genus, resulting in only 61.5% of the isolates correctly identified at species level while the correctness reached 94.6% at genus level. Using this extended in-house database, MALDI-TOF MS thus appears superior to morphology in order to obtain a robust and accurate identification of filamentous fungi. A continuous extension of the library is however necessary to further improve its reliability. Indeed, 15 isolates were still not represented while an additional three isolates were not recognized, probably because of a lack of intraspecific variability of the corresponding species in the database.

Identification, characterization, and expression levels of putative adhesive proteins from the tube-dwelling polychaete Sabellaria alveolata.

The shelter of the tube-dwelling polychaete Sabellaria alveolata is composed of mineral particles assembled with spots of a proteinaceous cement. The adhesive proteins constituting the cement were identified on the basis of their sequence similarity with proteins of a phylogenetically related species, Phragmatopoma californica. Two positively charged proteins, Sa-1 and Sa-2, share common features: they both have a mass of 22 kDa; are rich in glycine, tyrosine and basic residues; and show repeated peptide motifs. The consensus repeat of Sa-1 is KGAYGAKGLGYGNKAGYGAYG (occurring 6-8 times), while Sa-2 displays the consensus heptapeptide VHKAAWG (5 times) and undecapeptide VHKAAGYGGYG (8 times). Two variants of a serine-rich protein, Sa-3A (22 kDa) and Sa-3B (21 kDa), were also identified. Their serine residues account for 75 mol% and are probably phosphorylated, meaning that Sa-3 is very acidic and negatively charged. Moreover, tyrosine residues of all adhesive proteins are presumably modified into DOPA. Although protein sequences are not well-conserved between S. alveolata and P. californica, their main characteristics (including amino acid composition, post-translational modifications, repeated patterns, isoelectric point, and mass) are shared by both species. This suggests that these features are more important for their function than the primary structure of the proteins. The mRNA abundance for each protein was estimated by quantitative real-time PCR, revealing relative expression levels of about 5, 11, 1.5, and 1 for Sa-1, -2, -3A, and -3B, respectively. These levels could be indicative of charge neutralization phenomena or could reflect their function (interface vs. bulk) in the cement.

Characterization of the bacterial community associated with body wall lesions of Tripneustes gratilla (Echinoidea) using culture-independent methods.

The bacterial community associated with skin lesions of the sea urchin Tripneustes gratilla was investigated using 16S ribosomal RNA gene cloning and fluorescent in situ hybridization (FISH). All clones were classified in the Alphaproteobacteria, Gammaproteobacteria and Cytophaga-Flexibacter-Bacteroides (CFB) bacteria. Most of the Alphaproteobacteria were related to the Roseobacter lineage and to bacteria implicated in marine diseases. The majority of the Gammaproteobacteria were identified as Vibrio while CFB represented only 9% of the total clones. FISH analyses showed that Alphaproteobacteria, CFB bacteria and Gammaproteobacteria accounted respectively for 43%, 38% and 19% of the DAPI counts. The importance of the methods used is emphasized.

Characterization of the bacterial communities associated with the bald sea urchin disease of the echinoid Paracentrotus lividus.

The microbial communities involved in the bald sea urchin disease of the echinoid Paracentrotus lividus are investigated using culture-independent techniques. Lesions of diseased specimens from two locations in France, La Ciotat (Mediterranean Sea) and Morgat (Atlantic Ocean), are examined by Scanning Electron Microscopy (SEM) and the diversity of their microbiota is analysed by Denaturing Gradient Gel Electrophoresis (DGGE) and 16S rRNA gene clones libraries construction. Microscopic observations demonstrated that only the central area of the lesions is invaded by bacteria but not the peripheral zone and the surrounding healthy tissues. Molecular analysis identified at least 24 bacterial genomospecies in bald sea urchin lesions: 5 are Alphaproteobacteria, 10 are Gammaproteobacteria, 8 are CFB bacteria and 1 is a Fusobacteria. Out of them, 4 are observed in both locations while 10 occur only in the Atlantic Ocean and 10 only in the Mediterranean Sea. Gammaproteobacteria are the most represented in clones libraries from both locations, with respectively 65% and 43% of the total clones. CFB and Alphaproteobacteria accounted for the majority of the remaining clones and were detected by DGGE in virtually all samples from both stations. Our results demonstrate that bacterial communities observed on diseased individuals of the same echinoid species but originating from distinct locations are not similar and thus support the hypothesis that bacteria involved in the worldwide echinoid disease commonly called the bald sea urchin disease are opportunistic and not specific.