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Enterococcus is a controversial genus due to its great variability; this genus includes pathogenic strains, spoilage strains, and apparently safe strains including some probiotic strains. Previous studies focused on the characterization of strains of Enterococcus spp. involved in nosocomial infections. However, little research has been conducted on Enterococcus strains in foodstuffs. In the present work, 36 strains of different species of Enterococcus have been characterized by means of MALDI-TOF MS, resulting in highly specific mass spectral fingerprints. Characteristic peak masses common to certain bacterial species of Enterococcus have been identified. Thus, a peak at m/z 4426 ± 1 was assigned as a genus-specific biomarker. In addition, phyloproteomic relationships based on the mass spectral data were compared to the results of a phylogenetic analysis based on the 16S rRNA gene sequence. A better grouping at the species level was observed in the phyloproteomic tree, especially for the Enterococcus faecium group. Presumably, the assortment of some strains or ecotypes could be related to their ecological niche specialization. The approach described in this study leads the way toward the rapid and specific identification of different strains and species of Enterococcus in food based on molecular protein markers, aiming at the early detection of pathogenic strains and strains implicated in food poisoning or food spoilage., (© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The present study aims to compare two molecular technologies, 16S rRNA sequencing and MALDI-TOF MS, for bacterial species identification in seafood. With this aim, 70 reference strains from culture collections, including important seafood-borne pathogenic and spoilage bacterial species, and 50 strains isolated from commercial seafood products, were analysed by both techniques. Genomic analysis only identified the species of 50% of the isolated strains, proving to be particularly poor at identifying members of the Pseudomonas and Bacillus genera. In contrast, MALDI-TOF MS fingerprinting identified 76% of the strains at the species level. The mass spectral data were submitted to the SpectraBank database (http://www.spectrabank.org), making this information available to other researchers. Furthermore, cluster analysis of the peak mass lists was carried out with the web application SPECLUST and the calculated groupings were consistent with results determined by a phylogenetic approach that is based on the 16S rRNA sequences. However, the MALDI-TOF MS analysis demonstrated more discriminating potential that allowed for better classification, especially for the Pseudomonas and Bacillus genera. This is of importance with respect to the varying pathogenic and spoilage character at the intragenus and intraspecies level. In this sense, MALDI-TOF MS demonstrated to be a competent bacterial typing tool that extends phenotypic and genotypic approaches, allowing a more ample classification of bacterial strains., (© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Staphylococcus aureus is a known pathogen, causing serious food-borne intoxications due to the production of enterotoxins, being otherwise a major cause of mastitis. In this sense, the detection of S. aureus is an important issue for the food industry to avoid health hazards and economic losses. The present work applied MALDI-TOF MS for the classification of 40 S. aureus strains, 36 isolated from Italian dairy products and four from human samples. All isolated strains were clearly identified as S. aureus by their spectral fingerprints. The peak masses m/z 3444, 5031, and 6887 were determined to be specific biomarkers for S. aureus. Furthermore, clustering of the peak mass lists was successfully applied as a typing method, resulting in eight groups of strains. This is the first time that a detailed spectral comparison was carried out and characteristic peak masses were determined for every spectral group. Three strains exhibited a peak at m/z 6917 instead of m/z 6887, which was related to four polymorphisms in their 16S rRNA sequences. However, the grouping obtained by MALDI-TOF MS fingerprinting could not be related to toxin production or to the origin of the strains., (© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

MALDI-TOF MS has proved to be an accurate, rapid, and cost-effective technique for microbial identification in which the spectral fingerprint of an unknown strain can be compared to a database of spectra from reference strains. Most of the existing databases are private and often costly to access, and little spectral information is shared among researchers. The objective of the present communication is to introduce the SpectraBank database (http://www.spectrabank.org), which provides open access MALDI-TOF mass spectra from a variety of microorganisms. This work aims to familiarize readers with the SpectraBank database, from the sample preparation, data collection, and data analysis to how the spectral reference data can be used for microbial species identification. The database currently includes more than 200 MALDI-TOF MS spectra from more than 70 bacterial species and links to the freely available web-based application SPECLUST (http://bioinfo.thep.lu.se/speclust.html) to allow comparisons of the obtained peak mass lists and evaluate phyloproteomic relationships. The SpectraBank database is intended to be expanded by the addition of new spectra from microbial strains, obtained in our laboratory and by other researchers., (© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The rapid identification of food pathogenic and spoilage bacteria is important to ensure food quality and safety. Seafood contaminated with pathogenic bacteria is one of the major causes of food intoxications, and the rapid spoilage of seafood products results in high economic losses. In this study, a collection of the main seafood pathogenic and spoilage Gram-positive bacteria was compiled, including Bacillus spp., Listeria spp., Clostridium spp., Staphylococcus spp. and Carnobacterium spp. The strains, belonging to 20 different species, were obtained from the culture collections and studied by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). A reference library was created, including the spectral fingerprints of 32 reference strains and the extracted peak lists with 10-30 peak masses. Genus-specific as well as species-specific peak masses were assigned and could serve as biomarkers for the rapid bacterial identification. Furthermore, the peak mass lists were clustered with the web-application SPECLUST to show the phyloproteomic relationships among the studied strains. Afterwards, the method was successfully applied to identify six strains isolated from seafood by comparison with the reference library. Additionally, phylogenetic analysis based on the 16S rRNA gene was carried out and contrasted with the proteomic approach. This is the first time MALDI-TOF MS fingerprinting is applied to Gram-positive bacterial identification in seafood, being a fast and accurate technique to ensure seafood quality and safety., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Histamine poisoning is caused by the consumption of fish and other foods that harbor bacteria possessing histidine decarboxylase activity. With the aim of preventing histamine formation, highly specific mass spectral fingerprints were obtained from the 16 major biogenic amine-producing enteric and marine bacteria by means of MALDI-TOF MS analysis. All bacterial strains analyzed exhibited specific spectral fingerprints that enabled its unambiguous differentiation. This technique also identified peaks common to certain bacterial groups. Thus, two protein peaks at m/z 4182+/-1 and 8363+/-6 were found to be present in all Enterobacteriaceae species analyzed except for Morganella morganii. Peaks at m/z 3635+/-1 and 7267+/-2 were specific to both M. morganii and Proteus spp. Biogenic amine-forming Proteus spp. exhibited three genus-specific peaks at m/z 3980, 7960+/-1 and 9584+/-2. The genus Photobacterium also showed three genus-specific peaks at m/z 2980+/-1, 4275+/-1 and 6578+/-1. The two histamine-producing Gram-positive bacteria Lactobacillus sp. 30A and Staphylococcus xylosus exhibited a few protein peaks in the 2000-7000 m/z range and could be easily distinguished from biogenic amine-forming Gram-negative bacteria. Clustering based on MALDI-TOF MS also exhibited a good correlation with phylogenetic analysis based on the 16S rRNA gene sequence, validating the ability of the MALDI-TOF technique to establish relationships between microbial strains and species. The approach described in this study leads the way toward the rapid and specific identification of major biogenic amine-forming bacteria based on molecular protein markers with a goal to the timely prevention of histamine food poisoning.