The microbial spoilage of meat and seafood products with short shelf lives is responsible for a significant amount of food waste. Food spoilage is a very heterogeneous process, involving the growth of various, poorly characterized bacterial communities. In this study, we conducted 16S ribosomal RNA gene pyrosequencing on 160 samples of fresh and spoiled foods to comparatively explore the bacterial communities associated with four meat products and four seafood products that are among the most consumed food items in Europe. We show that fresh products are contaminated in part by a microbiota similar to that found on the skin and in the gut of animals. However, this animal-derived microbiota was less prevalent and less abundant than a core microbiota, psychrotrophic in nature, mainly originated from the environment (water reservoirs). We clearly show that this core community found on meat and seafood products is the main reservoir of spoilage bacteria. We also show that storage conditions exert strong selective pressure on the initial microbiota: alpha diversity in fresh samples was 189±58 operational taxonomic units (OTUs) but dropped to 27 ± 12 OTUs in spoiled samples. The OTU assemblage associated with spoilage was shaped by low storage temperatures, packaging and the nutritional value of the food matrix itself. These factors presumably act in tandem without any hierarchical pattern. Most notably, we were also able to identify putative new clades of dominant, previously undescribed bacteria occurring on spoiled seafood, a finding that emphasizes the importance of using culture-independent methods when studying food microbiota.

Lactic acid bacteria (LAB) isolated from fish products (fresh fish, smoked and marinated fish, fish intestinal tract) were screened for bacteriocin production and immunity in conditions eliminating the effects of organic acids and hydrogen peroxide. Twenty-two isolates which were found to produce bacteriocin-like compounds were identified as Carnobacteria, Lactococci and Enterococci on the basis of morphological examination, gas production from glucose, growth temperatures, configuration of lactic acid, carbohydrates fermentation and deamination of arginine. Two Carnobacteria named V1 and V41 were selected for further studies and identified by DNA-DNA hybridization as Carnobacterium piscicola and Carnobacterium divergens, respectively. Their respective bacteriocins named piscicocin V1 and divercin V41 were heat-resistant and sensitive to various proteolytic enzymes. These bacteriocins were active against Listeria monocytogenes and exhibited a different spectrum of activity against LAB. Both bacteriocins had a bactericidal and non-bacteriolytic mode of action. Maximum production of piscicocin V1 and divercin V41 in Man Rogosa Sharpe (MRS) medium broth occurred at the beginning of the stationary phase and was higher at 20°C than at 30°C. When the cultures were maintained at pH 6.5, bacteriocin production was significantly increased.

Divercin V41 is a new bacteriocin produced by Carnobacterium divergens V41, a lactic acid bacterium isolated from fish viscera. The amino acid sequence of divercin V41 showed high homologies with pediocin PA-1 and enterocin A. Two disulphide bonds were present in the hydrophilic N-terminal domain and in the highly variable hydrophobic C-terminal domain, respectively. A DNA probe designed from the N-terminal sequence of the purified peptide was used t o locate the structural gene of divercin V41. A 6 kb chromosomal fragment containing the divercin V41 structural gene (dvnA) was cloned and sequenced. The results indicate that divercin V41 is synthesized as a pre-bacteriocin of 66 amino acids. The 23-residue N-terminal extension is cleaved off t o yield the mature 43-amino-acid divercin V41. In addition, the fragment encodes putative proteins commonly found within bacteriocin operons, including an ATPdependent transporter, two immunity-like proteins and the two components of a lantibiotic-type signal-transducing system. The genetic organization of the fragment suggested important gene rearrangements.

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The bacteriocin-producing strain Enterococcus faecium ST5Ha was isolated from smoked salmon and identified by biomolecular techniques. Ent. faecium ST5Ha produces a pediocin-like bacteriocin with activity against several lactic acid bacteria, Listeria spp. and some other human and food pathogens, and remarkably against HSV-1 virus. Bacteriocin ST5Ha was produced at high levels in MRS broth at 30 degrees C and 37 degrees C, reaching a maximum production of 1.0 x 10(9) AU/ml, checked against Listeria ivanovii ATCC19119 as target strain and surrogate of pathogenic strain Listeria monocytogenes. The molecular weight of bacteriocin ST5Ha was estimated to be 4.5 kDa according to tricine-SDS-PAGE data. Ent. faecium ST5Ha harbors a 1.044 kb chromosomal DNA fragment fitting in size to that of pediocin PA-1/AcH. In addition, the sequencing of bacteriocin ST5Ha gene indicated 99% of DNA homology to pediocin PA-1/AcH. The combined application of low levels (below MIC) of ciprofloxacin and bacteriocin ST5Ha resulted in a synergetic effect in the inhibition of target strain L. ivanovii ATCC19119. Bacteriocin ST5Ha displayed antiviral activity against HSV-1, an important human pathogen, with a selectivity index of 173. To the best of our knowledge, this is the first report on Ent. faecium as a potential producer of pediocin-like bacteriocin with antiviral activity.

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Screening of a library of Enterococcus faecalis insertional mutants allowed isolation of a mutant affected in tyramine production. The growth of this mutant was similar to that of the wild-type E. faecalis JH2-2 strain in Maijala broth, whereas high-performance liquid chromatography analyses showed that tyramine production, which reached 1,000 g ml ؊1 for the wild-type strain, was completely abolished. Genetic analysis of the insertion locus revealed a gene encoding a decarboxylase with similarity to eukaryotic tyrosine decarboxylases. Sequence analysis revealed a pyridoxal phosphate binding site, indicating that this enzyme belongs to the family of amino acid decarboxylases using this cofactor. Reverse transcription-PCR analyses demonstrated that the gene (tdc) encoding the putative tyrosine decarboxylase of E. faecalis JH2-2 is cotranscribed with the downstream gene encoding a putative tyrosine-tyramine antiporter and with the upstream tyrosyl-tRNA synthetase gene. This study is the first description of a tyrosine decarboxylase gene in prokaryotes.Biogenic amines in food result mainly from microbial activity due to amino acid decarboxylation (16,49). Histamine and tyramine have been the most studied biogenic amines due to the toxicological effects derived from their vasoactive and psychoactive properties. Histamine has been recognized as the causative agent of scromboid poisoning (histamine intoxication), whereas tyramine has been related to food-induced migraines and hypertensive crisis (39). Various tyramine concentrations have been found in many foods, including cheeses, drinks, and meat and fish products (30,44,45), and a dose of only 6 mg total tyramine intake may be dangerous for patients under antidepressive treatment who are receiving monoamine oxidase inhibitors (42). The formation of tyramine in foods depends on the concentration of free tyrosine and the presence of microorganisms having tyrosine decarboxylase activity. Many microorganisms could be implicated in tyramine production. For example, some bacteria belonging to the genera Enterococcus, Carnobacterium, and Lactobacillus have been found to be tyramine producers (5, 31, 35). However, while tyrosine decarboxylase enzymes have been well characterized in eukaryotes, for example, in parsley (Petroselinum crispum) and in Drosophila (24, 47), little is known about tyrosine decarboxylase in prokaryotes. Indeed, only a few reports have described physiological studies of the influence of some physicochemical factors, such as temperature, pH, NaCl, or tyrosine concentration, on tyramine production by Lactobacillus curvatus (46), Lactobacillus brevis (35), and Carnobacterium divergens (32). Tyrosine decarboxylase purification and characterization of the enzyme have been reported only for Enterococcus faecalis (previously called Streptococcus faecalis) (4) and for L. brevis IOEB 9809 and ATCC 367 (34, 36). These authors have shown that tyrosine decarboxylases in E. faecalis and L. brevis have an [␣2] dimmer structure with two subunits of approximately...