Species Identification of Food Spoilage and Pathogenic Bacteria by MALDI-TOF Mass Fingerprinting

18 pages, 1 table, 2 figuresFood quality and safety is an increasingly important public health issue. Nowadays, the topics “food quality” and “food safety” are very close and two important issues in the food sector, due to the globalization of the food supply and the increased complexity of the food chain. The consumers need to purchase safe products that do not involve any kind of risk for health. On one hand, the aim of the “food safety” is to avoid health hazards for the consumer: microbiological hazards, pesticide residues, misuse of food additives and contaminants, such as chemicals, biological toxins and adulteration. On the other hand, “food quality” includes all attributes that influence the value of a product for the consumer; this includes negative attributes such as spoilage, contamination with filth, discoloration, off-odors and positive attributes such as the origin, color, flavor, texture and processing method of the food (FAO, 2003)This work was funded by project 10PXIB261045PR from Xunta de Galicia and by project AGL2010-19646 from the Spanish Ministry of Science and Technology. The work of K. Böhme and I.C. Fernández-No is supported by a “Maria Barbeito” and “Lucas Labrada” research contract from Xunta de GaliciaN

Caracterización de Staphylococcus aureus aislado de productos lácteos por MALDI-TOF MS

Comunicaciones a congreso

SpectraBank: An open access tool for rapid microorganism identification by MALDI-TOF MS fingerprinting

13 páginas, 3 figurasMatrix-assisted laser desorption/ionisation (MALDI) time-of-flight (TOF) mass spectrometry (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 familiarise 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.This work was funded by project 10PXIB261045PR from Xunta de Galicia and by project AGL2010- 19646 from the Spanish Ministry of Science and Technology.Peer reviewe

Comparative analysis of protein extraction methods for the identification of seafood-borne pathogenic and spoilage bacteria by MALDI-TOF mass spectrometry

7 páginas, 4 figuras, 2 tablasSpecies differentiation of food pathogenic and spoilage bacteria is important to ensure food quality and safety. Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has been applied to species identification of microorganisms, proving to be a rapid and costeffective technique and allowing species differentiation due to the highly specific spectral profiles obtained. In this work, bacterial strains from our laboratory intern collection of seafood-borne pathogenic and spoilage species were studied by MALDI-TOF MS. Different sample preparation protocols were applied and compared to each other. Two methods were based on the analysis of whole bacterial cells that were suspended in an organic solvent or applied directly to the sample target. In a different sample preparation technique, cell extracts were obtained from intact bacterial cells by a dissolution/centrifugation step. The protocol applied for the study of cell extracts was shown to be very fast and simple, allowing the standardization of sample preparation. Furthermore, the analysis of cell extracts had several advantages with respect to the analysis of suspensions of whole bacterial cells. Thus, spectral profiles obtained from cell extracts showed less noise and more reproducible peaks as compared to spectra obtained from intact cells. The analysis of cell extracts by MALDI-TOF MS was also applied to create a mass spectral library of the main pathogenic and spoilage bacteria potentially present in seafood and was demonstrated to be a rapid and accurate method for microbial species differentiation, as well as for the classification of unknown strains isolated from seafood.This work was funded by the PGIDIT Research Program (Project PGIDIT06PXIB261164PR) of the Xunta de Galicia (Galician Council for Industry Commerce and Innovation). The work of K. Bohme and I.C. Fernandez-No is supported by a ‘‘Maria Barbeito’’ and ‘‘Lucas Labrada’’ research contract from Xunta de Galicia.Peer reviewe

Utilisation d'acides lactiques et citriques dans la glace afin d'améliorer la qualité de deux espèces de poissons pendant l'entreposage frigorifique à bord des navires de pêche

8 páginas, 3 tablas, 3 figurasThis work focused on the on-board chilled storage of European hake (Merluccius merluccius) and megrim (Lepidorhombus whiffiagonis). To enhance fish quality, an aqueous solution including citric (1.25 g l−1) and lactic (0.50 g l−1) acids was prepared, frozen, ground and employed as icing medium. Its effect on sensory, microbiological and chemical changes was monitored after 9, 12 and 15 days of on-board storage. Lower (p < 0.05) bacterial growth was detected according to microbiological (aerobe, anaerobe, psychrotrophe, proteolytic, and Enterobacteriaceae counts) and chemical (trimethylamine content) assessments. An inhibitory effect (p < 0.05) on autolysis development (K value assessment) in hake was also detected. Finally, an enhancement of sensory scores (eyes, external odour and gills) in both species was obtained. Results described allow to conclude that on-board employment of such acid-mixture icing system can provide a profitable strategy to obtain higher quality and safe products while unloadingThis work was supported by the Secretaría Xeral de I+D from the Xunta de Galicia (Galicia, Spain) through the Research Project 10 TAL 018 EPeer reviewe

Safety assessment of fresh and processed seafood products by MALDI-TOF mass fingerprinting

12 páginas, 4 figuras, 3 tablas.-- The original publication is available at www.springerlink.comFoodborne intoxications caused by the consumption of fish and other products of marine origin contaminated with bacterial pathogens are an ever-present threat, either due to bacteria and/or its metabolites. In addition, the rapid spoilage of seafood due to microbial activity, results in high economic losses. The development of the microbiota in seafood products depends on the microbiological ambience of capture, processing and storage, and the applied preservation method. Thus, pathogenic and spoilage bacterial species in seafood may come from the indigenous microbiota of the aquatic ambience or are introduced by contamination during processing. Rapid and accurate bacterial species identification is essential for an effective control program to ensure safety and quality of either processed or minimally processed seafood. In the present work, matrix-assisted laser desorption ionizationtime of flight mass spectrometry was successfully applied to identify 26 bacterial strains isolated from fresh fish and processed seafood samples. The approach was based on the comparison of unknown spectra to a reference spectral library and demonstrated to be a fast and accurate technique for bacterial species differentiation, which can be used for the rapid identification of foodborne pathogens and spoilage bacteria potentially present in products of marine origin.This work was funded by the PGIDIT Research Program (Project PGIDIT06PXIB261164PR) of the Xunta de Galicia (Galician Council for Industry Commerce and Innovation). The work of K. Bohme and I.C. Fernandez-No is supported by a “Maria Barbeito” and “Lucas Labrada” research contract from Xunta de Galicia.Peer reviewe

Rapid species identification of seafood spoilage and pathogenic Gram-positive bacteria by MALDI-TOF mass fingerprinting

15 páginas, 4 tablas, 6 figurasThe 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 matrixassisted 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 speciesspecific 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 Grampositive bacterial identification in seafood, being a fast and accurate technique to ensure seafood quality and safety.This work was funded by project 10PXIB261045PR from Xunta de Galicia and by project AGL2010-19646 from the Spanish Ministry of Science and Technology. The work of K. Bohme and I. C. Fernandez-No is supported by a ‘Maria Barbeito’ and ‘Lucas Labrada’ research contract from Xunta de Galicia.Peer reviewe

Evaluation of a novel 16S rRNA/tRNAVal mitochondrial marker for the identification and phylogenetic analysis of shrimp species belonging to the superfamily Penaeoidea

8 páginas, 4 figuras, 5 tablasIn this study, we infer the phylogenetic relationships within commercial shrimp using sequence data from a novel mitochondrial marker consisting of an approximately 530-bp region of the 16S ribosomal RNA (rRNA)/transfer RNA (tRNA)Val genes compared with two other mitochondrial genes: 16S rRNA and cytochrome c oxidase I (COI). All three mitochondrial markers were considerably AT rich, exhibiting values up to 78.2% for the species Penaeus monodon in the 16S rRNA/tRNAVal genes, notably higher than the average among other Malacostracan mitochondrial genomes. Unlike the 16S rRNA and COI genes, the 16S rRNA/tRNAVal marker evidenced that Parapenaeus is more closely related to Metapenaeus than to Solenocera, a result that seems to be more in agreement with the taxonomic status of these genera. To our knowledge, our study using the 16S rRNA/tRNAVal gene as a marker for phylogenetic analysis offers the first genetic evidence to confirm that Pleoticus muelleri and Solenocera agassizi constitute a separate group and that they are more related to each other than to genera belonging to the family Penaeidae. The 16S rRNA/tRNAVal region was also found to contain more variable sites (56%) than the other two regions studied (33.4% for the 16S rRNA region and 42.7% for the COI region). The presence of more variable sites in the 16S rRNA/tRNAVal marker allowed the interspecific differentiation of all 19 species examined. This is especially useful at the commercial level for the identification of a large number of shrimp species, particularly when the lack of morphological characteristics prevents their differentiation.The authors acknowledge the financial support from the National Food Program of the INIA (Spanish Ministry for Education, project CAL-03-030-C2-1) and from the PGIDIT Research Program in Marine Resources (project PGIDIT04RMA261004PR) of the Xunta de Galicia (Galician Council for Industry Commerce and Innovation).Peer reviewe

Isolation and characterization of Streptococcus parauberis from vacuum-packaging refrigerated seafood products

7 páginas, 3 figuras, 4 tablasStreptococcus parauberis is known as an etiological agent of mastitis in cows and for producing streptococcosis in farmed fish, although its presence in foods has seldom been reported. In this work, two bacterial isolates were recovered from a spoiled vacuum-packaged refrigerated seafood product. Both isolates were identified by 16S rRNA gene sequencing, exhibiting 99% homology with respect to S. parauberis. Both isolates were also characterized by Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS). Genetic analysis revealed the clonal homogeneity of the isolates and their grouping together with other S. parauberis strains in a different cluster with respect to Streptococcus uberis strains. Proteomic analysis by MALDI-TOF MS allowed for the identification of five mass peaks in the range of 2200–6000 m/z that resulted to be specific to the species S. parauberis and allowed its rapid and direct identification with respect to other pathogenic and spoilage bacteria potentially present in seafood and other food products. This study represents, to our knowledge, the first report of S. parauberis in seafood in general and in vacuum-packed food products in particular. Moreover, it provides a rapid method based on MALDI-TOF MS for the identification of S. parauberis.This work was funded by project 10PXIB261045PR from Xunta de Galicia and by project AGL2010-19646 from the Spanish Ministry of Science and Technology.Peer reviewe

Species differentiation of seafood spoilage and pathogenic gram-negative bacteria by MALDI-TOF mass fingerprinting

15 páginas, 7 figuras, 6 tablasSpecies differentiation is important for the early detection and identification of pathogenic and food-spoilage microorganisms that may be present in fish and seafood products. The main 26 species of seafood spoilage and pathogenic Gram-negative bacteria, including Aeromonas hydrophila, Acinetobacter baumanii, Pseudomonas spp., and Enterobacter spp. among others, were characterized by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) of low molecular weight proteins extracted from intact bacterial cells by a fast procedure. From the acquired spectra, a library of specific mass spectral fingerprints was constructed. To analyze spectral fingerprints, peaks in the mass range of 2000−10 000 Da were considered and representative mass lists of 10−35 peak masses were compiled. At least one unique biomarker peak was observed for each species, and various genus-specific peaks were detected for genera Proteus, Providencia, Pseudomonas, Serratia, Shewanella, and Vibrio. Phyloproteomic relationships based on these data were compared to phylogenetic analysis based on the 16S rRNA gene, and a similar clustering was found. The method was also successfully applied for the identification of three bacterial strains isolated from seafood by comparing the spectral fingerprints with the created library of reference fingerprints. Thus, the proteomic approach demonstrated to be a competent tool for species identificationThis work was funded by the PGIDIT Research Program (Project PGIDIT06PXIB261164PR) of the Xunta de Galicia (Galician Council for Industry Commerce and Innovation).Peer reviewe