Xavier Dousset

Evidence for two bacteriocins produced by Carnobacterium piscicola and Carnobacterium divergens isolated from fish and active against Listeria monocytogenes

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.

Detection and characterization of a novel antibacterial substance produced by Lactobacillus plantarum ST 31 isolated from sourdough.

Lactobacillus plantarum ST31 isolated from sourdough produced an antimicrobial substance inhibiting other strains of the genera Lactobacillus, Leuconostoc, Pediococcus, Streptococcus, Bacillus and some foodborne pathogens including Staphylococcus aureus. This antimicrobial substance was inactivated by proteolytic enzymes. Consequently, it was characterized as a bacteriocin and was designated plantaricin ST31. This bacteriocin was stable in the pH range 3-8 and it was not affected by amylolytic enzymes. Production of plantaricin was pH and temperature dependent, and maximum yields were obtained in MRS broth cultures maintained at pH 6 and incubated at 30 degrees C in the exponential phase to the early stationary growth phase of the producer organism. This bacteriocin was purified by using consecutive ammonium sulfate and reversed-phase chromatography. It is a peptide of 20 amino acid residues with a mass of 2755+/-0.3 Da, as determined by electrospray mass spectrometry. The sequence of Plantaricin ST31 showed no similarity to those of other bacteriocins. Plantaricin ST31 production appeared to be chromosomally encoded.

Inhibition of Listeria monocytogenes by in situ produced and semipurified bacteriocins of Carnobacterium spp. on vacuum-packed, refrigerated cold-smoked salmon.

Listeria monocytogenes inhibition by Carnobacterium strains and crude bacteriocins on sterile and commercial vacuum-packed cold-smoked salmon stored at 4 degrees C and 8 degrees C was investigated. Carnobacterium piscicola V1 was bactericidal against L. monocytogenes at the two temperatures, whereas Carnobacterium divergens V41 presented a bacteriostatic effect. C. piscicola SF668 delayed L. monocytogenes growth at 8 degrees C and had a bacteriostatic effect at 4 degrees C. Listeria growth was not affected by a non-bacteriocin-producing C. piscicola. Crude extracts of piscicocins were bactericidal at 4 degrees C and 8 degrees C. Listeria growth was delayed by divercin V41 at 8 degrees C and was inhibited at 4 degrees C. Nisin delayed Listeria growth at 8 degrees C and was bacteriostatic at 4 degrees C. The present study demonstrates that L. monocytogenes growth could be prevented on vacuum-packed cold-smoked salmon by Carnobacterium and associated bacteriocins at chilled temperatures. Moreover, no product spoilage could be observed with the use of such bacteriocin-producing strains as demonstrated by good sensorial analyses and low biogenic amine production.

Isolation and partial biochemical characterization of a proteinaceous anti-bacteria and anti-yeast compound produced by Lactobacillus paracasei subsp. paracasei strain M3.

New proteinaceous active substance produced by Lactobacillus paracasei subsp. paracasei strain M3 used as a starter for Bulgarian yellow cheese was identified and studied. It displayed bactericidal and fungistatic activities. Its activity was checked against over 60 bacterial and yeast strains. It was efficient against Bacillus subtilis ATCC 6633, several L. delbrueckii species, Helicobacter pylori NCIPD 230 and some yeast species, for example Candida albicans, C. pseudointermedia NBIMCC 1532, C. blankii NBIMCC 85 and Saccharomyces cerevisiae NBIMCC 1812. The synthesis of the substance by producing strain was detected in the late logarithmic growth phase during batch fermentation. Anion exchange chromatography, reversed phase chromatography (RPC) on C4 column and HPLC on C18 column were used for partial purification of this antimicrobial compound. The gene responsible for the synthesis of the active substance is located on the bacterial chromosome.

Inhibition of Listeria monocytogenes by Carnobacterium spp. strains in a simulated cold smoked fish system stored at 4°C

Preservation of smoked salmon from bacterial spoilage, and especially from Listeria monocytogenes, by bacteriocin producers is a promising challenge. Over a hundred lactic acid bacteria, isolated from commercial vacuum packaged cold smoked salmon, were screened for their antagonistic activity against L. innocua. Twenty-two strains were able to produce bacteriocin-like proteinaceous substances. These strains were characterized physiologically and biochemically as Carnobacterium strains. Three different groups were determined by pulsed-field gel electrophoresis after Sma I and Apa I DNA digestion. Peptidoglycan hydrolases patterns completed the characterization of these strains. All were confirmed as being Carnobacterium piscicola. Growth and bacteriocin production of three strains of each group and two well known bacteriocin producers (C. divergens V41 and C. piscicola V1) were tested in a simulated cold smoked fish system at 4°C. These strains were able to reach 108 cfu ml−1 in 21 days and to produce as much bacteriocin activities in the cold smoked fish system as in the rich media. Carnobacterium divergens V41 and C. piscicola V1 were the most effective strains in co-culture experiments, inhibiting L. monocytogenes as early as day 4, whereas C. piscicola SF668 inhibiting effect was observed at day 13. The potential for using such biopreservation treatments on whole smoked salmon is discussed.

Identification of the Enterococcus faecalis Tyrosine Decarboxylase Operon Involved in Tyramine Production

ABSTRACT 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 μg 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.

Origin and ecological selection of core and food-specific bacterial communities associated with meat and seafood spoilage

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.

Lactobacillus salivarius: Bacteriocin and probiotic activity

Lactic acid bacteria (LAB) antimicrobial peptides typically exhibit antibacterial activity against food-borne pathogens, as well as spoilage bacteria. Therefore, they have attracted the greatest attention as tools for food biopreservation. In some countries LAB are already extensively used as probiotics in food processing and preservation. LAB derived bacteriocins have been utilized as oral, topical antibiotics or disinfectants. Lactobacillus salivarius is a promising probiotic candidate commonly isolated from human, porcine, and avian gastrointestinal tracts (GIT), many of which are producers of unmodified bacteriocins of sub-classes IIa, IIb and IId. It is a well-characterized bacteriocin producer and probiotic organism. Bacteriocins may facilitate the introduction of a producer into an established niche, directly inhibit the invasion of competing strains or pathogens, or modulate the composition of the microbiota and influence the host immune system. This review gives an up-to-date overview of all L. salivarius strains, isolated from different origins, known as bacteriocin producing and/or potential probiotic.

Sensory characteristics of spoilage and volatile compounds associated with bacteria isolated from cooked and peeled tropical shrimps using SPME-GC-MS analysis

The spoilage potential of six bacterial species isolated from cooked and peeled tropical shrimps (Brochothrix thermosphacta, Serratia liquefaciens-like, Carnobacterium maltaromaticum, Carnobacterium divergens, Carnobacterium alterfunditum-like and Vagococcus penaei sp. nov.) was evaluated. The bacteria were inoculated into shrimps, packaged in a modified atmosphere and stored for 27 days at 8 °C. Twice a week, microbial growth, as well as chemical and sensory changes, were monitored during the storage period. The bacteria mainly involved in shrimp spoilage were B. thermosphacta, S. liquefaciens-like and C. maltaromaticum whose main characteristic odours were cheese-sour, cabbage-amine and cheese-sour-butter, respectively. The volatile fraction of the inoculated shrimp samples was analysed by solid-phase microextraction (SPME) and gas chromatography coupled to mass spectrometry (GC-MS). This method showed that the characteristic odours were most likely induced by the production of volatile compounds such as 3-methyl-1-butanal, 2,3-butanedione, 2-methyl-1-butanal, 2,3-heptanedione and trimethylamine.

Study of the bacterial ecosystem in tropical cooked and peeled shrimps using a polyphasic approach.

The characterization of the microbial ecosystem of cooked tropical shrimps was carried out using a polyphasic approach. First, culture-dependent methods were used for bacterial enumeration and the phenotypic and molecular identification of bacterial isolates. Then, culture-independent methods, including PCR-TTGE (V3 region of the 16S rRNA gene), provided a fingerprinting of bacterial DNA directly extracted from shrimps. Two batches of cooked and peeled tropical shrimps were stored at 5 and 15 degrees C for 5 and 3 weeks, respectively. Trained panelists carried out a sensory evaluation and microbiological enumerations were performed. When spoilage of samples was perceived, several colonies were isolated from the total viable count media. Thus, 137 bacterial strains were identified by phenotypic and molecular tests. Lactic acid bacteria (LAB) constituted the major group with the most represented genera being Carnobacterium (C. divergens, C. maltaromaticum and indiscernible C. alterfunditum/pleistocenium), Vagococcus (indiscernible V. carniphilus/fluvialis) and Enterococcus (E. faecalis and E. faecium). The other groups corresponded to Brochothrix thermosphacta and Enterobacteriaceae (Serratia liquefaciens). In PCR-TTGE profiles some of DNA fragments were assigned to those of standard strains (S. liquefaciens, B. thermosphacta, E. faecalis, C. divergens and C. maltaromaticum) or identified isolates from culture-dependent analysis (E. faecium). Other additional informations were provided by fragment cloning (Psychrobacter sp, Citrobacter gillenii and Firmicute). In conclusion, TTGE is an excellent tool to monitor the evolution of the microbial ecosystem in seafood products.