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

Rapid proteomic characterization of bacteriocin-producing Enterococcus faecium strains from foodstuffs

Enterococcus belongs to a group of microorganisms known as lactic acid bacteria (LAB), which constitute a broad heterogeneous group of generally food-grade microorganisms historically used in food preservation. Enterococci live as commensals of the gastrointestinal tract of warm-blooded animals, although they also are present in food of animal origin (milk, cheese, fermented sausages), vegetables, and plant materials because of their ability to survive heat treatments and adverse environmental conditions. The biotechnological traits of enterococci can be applied in the food industry; however, the emergence of enterococci as a cause of nosocomial infections makes their food status uncertain. Recent advances in high-throughput sequencing allow the subtyping of bacterial pathogens, but it cannot reflect the temporal dynamics and functional activities of microbiomes or bacterial isolates. Moreover, genetic analysis is based on sequence homologies, inferring functions from databases. Here, we used an end-to-end proteomic workflow to rapidly characterize two bacteriocin-producing Enterococcus faecium (Efm) strains. The proteome analysis was performed with liquid chromatography coupled to a trapped ion mobility spectrometry-time-of-flight mass spectrometry instrument (TimsTOF) for high-throughput and high-resolution characterization of bacterial proteins. Thus, we identified almost half of the proteins predicted in the bacterial genomes (>1100 unique proteins per isolate), including quantifying proteins conferring resistance to antibiotics, heavy metals, virulence factors, and bacteriocins. The obtained proteomes were annotated according to function, resulting in 22 complete KEGG metabolic pathway modules for both strains. The workflow used here successfully characterized these bacterial isolates and showed great promise for determining and optimizing the bioengineering and biotechnology properties of other LAB strains in the food industry

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

Comunicaciones a congreso

Proteomic Characterization of Antibiotic Resistance, and Production of Antimicrobial and Virulence Factors in Streptococcus Species Associated with Bovine Mastitis. Could Enzybiotics Represent Novel Therapeutic Agents Against These Pathogens?

Streptococcus spp. are major mastitis pathogens present in dairy products, which produce a variety of virulence factors that are involved in streptococcal pathogenicity. These include neuraminidase, pyrogenic exotoxin, and M protein, and in addition they might produce bacteriocins and antibiotic-resistance proteins. Unjustifiable misuse of antimicrobials has led to an increase in antibiotic-resistant bacteria present in foodstuffs. Identification of the mastitis-causing bacterial strain, as well as determining its antibiotic resistance and sensitivity is crucial for effective therapy. The present work focused on the LC–ESI–MS/MS (liquid chromatography–electrospray ionization tandem mass spectrometry) analysis of tryptic digestion peptides from mastitis-causing Streptococcus spp. isolated from milk. A total of 2706 non-redundant peptides belonging to 2510 proteins was identified and analyzed. Among them, 168 peptides were determined, representing proteins that act as virulence factors, toxins, anti-toxins, provide resistance to antibiotics that are associated with the production of lantibiotic-related compounds, or play a role in the resistance to toxic substances. Protein comparisons with the NCBI database allowed the identification of 134 peptides as specific to Streptococcus spp., while two peptides (EATGNQNISPNLTISNAQLNLEDKNK and DLWC*NM*IIAAK) were found to be species-specific to Streptococcus dysgalactiae. This proteomic repository might be useful for further studies and research work, as well as for the development of new therapeutics for the mastitis-causing Streptococcus strainsThis work was supported by the Xunta de Galicia; the European Union Social Fund (ESF); the Spanish Ministry of Economy and Competitiveness (Project AGL 2.013-48.244-R); and by the European Regional Development Fund (ERDF) (2007-2013)S

Caracterización molecular de bacterias ácido-lácticas aisladas a partir de carne de ternera envasada al vacío de modo tradicional y mediante un sistema avanzado

A total of 91 lactic acid bacteria (LAB) were isolated from 50 beef samples, 25 packaged by traditional vacuum packaging and 25 packaged using advanced vacuum skin packaging. The isolated LAB were identified using 16S rRNA sequencing, whereas randomly amplified polymorphic DNA (RAPD-PCR) and cluster analysis were used for typing the LABs. Ten different species of LAB were identified and assigned to the following species: Enterococcus gilvus (22 isolates), Enterococcus faecium (9 isolates), Enterococcus casseliflavus (8 isolates), Enterococcus faecalis (4 isolates), Enterococcus malodoratus (3 isolates), Enterococcus devriessei (3 isolates), Lactobacillus sakei (15 isolates), Carnobacterium divergens (12 isolates), Carnobacterium maltaromaticum (5 isolates), and Leuconostoc mesenteroides (8 isolates). The RAPD profile bands observed were not significantly discriminatory, with exceptions of E. casseliflavus, E. faecalis, and E. faecium, which suggests that the type of packaging system used had no specific effect on the selection of most microbiota in the meat after packagingUn total de 91 bacterias ácido-lácticas (LAB) fueron aisladas a partir de 50 muestras de carne de ternera, de las cuales 25 fueron envasadas al vacío de modo tradicional y 25 fueron envasadas mediante un sistema de vacío avanzado. Los aislamientos fueron identificados a través de la secuenciación del ADN ribosomal 16S, mientras que la caracterización de las LAB se realizó mediante la amplificación al azar del ADN polimórfico (RAPD-PCR) y su posterior análisis cluster. Fueron identificadas cepas pertenecientes a diez especies bacterianas diferentes, en concreto: Enterococcus gilvus (22 aislamientos), E. faecium (9 aislamientos), E. casseliflavus (8 aislamientos), E. faecalis (4 aislamientos), E. malodoratus (3 aislamientos), E. devriessei (3 aislamientos), Lactobacillus sakei (15 aislamientos), Carnobacterium divergens (12 aislamientos), C. maltaromaticum (5 aislamientos) y Leuconostoc mesenteroides (8 aislamientos). Los perfiles de bandas de ADN obtenidas no revelaron diferencias significativas, con las excepciones de E. casseliflavus, E. faecalis y E. faecium, sugiriendo que el tipo de envasado no tiene un efecto específico en la selección de la mayor parte de la microbiota presente en la carne envasadaS

Enhanced quality and safety during on-board chilled storage of fish species captured in the Grand Sole North Atlantic fishing bank

8 páginas, 5 tablas, 3 figurasThe Grand Sole North Atlantic fishing bank is exploited by several European countries, although time lapsed between catch and destiny arrival can attain 15 days. In the present work, the use of slurry ice (SI) system was investigated for the on-board storage of chilled fish and carried out in parallel to traditional flake icing (FI). Three species (hake, Merluccius merluccius; angler, Lophius piscatorius; ray, Raja clavata) widely present in the mentioned bank were studied. A lower (p < 0.05) microbial (aerobes, psychrotrophes, proteolytics) development was observed in fishes subjected to SI system than in their counterpart specimens stored under FI. This correlated with lower (p < 0.05) productions of trimethylamine (hake and angler) and total volatile bases (ray) and extended shelf-life for fish species kept under SI conditions. In summary, on-board employment of SI can provide higher quality and safety products to consumer and allow increased commercial values while unloading and sale.This work was supported through a project grant by the Secretarı ´a Xeral de I+D from the Xunta de Galicia (Project PGIDIT 04 TAL 015E).Peer reviewe

Extension of the shelf life of chilled hake (Merluccius merluccius) by a novel icing medium containing natural organic acids

8 páginas, 4 tablas, 3 figurasThree combinations of two natural organic acids, citric acid (CA) and lactic acid (LA), in the form of novel flake icing systems were evaluated for the preservation of European hake (Merluccius merluccius), one of the most commercialised gadoid fish species in Europe. The flake icing systems were prepared with fresh water and 0.075%/0.050% (C-75 batch), 0.125%/0.050% (C-125 batch) and 0.175%/0.050% (C-175 batch) CA/LA, respectively. Aerobic mesophiles, psychrotrophs, proteolytic bacteria, anaerobes, Enterobacteriaceae, trimethylamine, lipid hydrolysis, primary, secondary and tertiary lipid oxidation events, and pH levels were evaluated in hake muscle in all batches during 13 d of chilled storage and the results were compared with sensory analyses. Significantly (p < 0.05) lower microbial counts were found in the C-175 batch for all five microbial groups investigated when compared with the control batch, with differences between batches of greater than 2 log CFU/g in the case of aerobes and anaerobes. The presence of organic acids in the icing systems also inhibited (p < 0.05) the increase in trimethylamine-nitrogen (TMA-N) content, with greater inhibition as the CA concentration in the ice increased. An inhibitory effect (p < 0.05) on fluorescent compound formation due to the presence of organic acids in the icing systems, which is indicative of tertiary lipid oxidation, was also observed at advanced storage times. These results correlated well with sensory analysis, which revealed that the shelf lives of the C-125 and C-175 batches were extended. The melting of the ice crystals containing the natural organic acid solutions, especially in the C-175 batch, exerted an antimicrobial washing effect that may explain the better protection of hake quality when stored in the proposed flake icing system.This work was supported by the Secretaría Xeral de I + D from the Xunta de Galicia (Galicia, Spain) through the Research Project 10 TAL 018E.Peer reviewe

Identification of commercial prawn and shrimp species of food interest by native isoelectric focusing

6 páginas, 3 figuras, 3 tablasNative isoelectric focusing (IEF) of water-soluble sarcoplasmic proteins was applied to the identification of 14 shrimp species of food interest belonging to the order Decapoda. These species have different commercial values, but due to their phenotypic similarities and carapace removal in their industrial processing, incorrect food labelling and deliberate or inadvertent adulteration can occur. Each of the 14 tested species showed species-specific protein band profiles and intra-specific polymorphism was low, not preventing the correct identification of the species. Therefore, IEF of water-soluble sarcoplasmic proteins allowed the differentiation of the 14 species considered. In addition, sarcoplasmic calcium-binding proteins (SCPs) were identified by tandem mass spectrometry (MS/MS) as the major species-specific proteins in these species, opening the way to further studies focusing on their potential use as specific biomarkersThis work was supported by the National Food Program of the INIA (Spanish Ministry for Education and Science) (Project CAL-03-030-C2-2) and by the PGIDIT Research Program in Marine Resources (Project PGIDIT04RMA261004PR) of the Xunta de Galicia (Galician Council for Industry, Commerce and Innovation). I.O. is supported by the FPU program (AP-2004-5826) and B.C. is supported by the RyC program, under the auspices of the Spanish MICINN.Peer reviewe

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