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International audience; The use of high-throughput methods allows a better characterization of food-related bacterial communities. However, such methods require large amounts of high-quality bacterial DNA, which may be a challenge when dealing with a complex matrix that has a low concentration of bacteria, such as fresh fish fillets. Therefore, the choice of method used to recover bacteria from a food matrix in a cost-effective way is critical, yet little information is available on the performance of commonly used methods. We assessed the recovery capacity of two such methods: stomaching and mechanical rinsing. The efficiency of the methods was evaluated through quantitative recovery and compatibility with end-point quantitative PCR (qPCR). Fresh rainbow trout (Oncorhynchus mykiss) fillets were inoculated with a bacterial marker, Brochothrix thermosphacta, at different concentrations (7.52 to 1.52 log CFU/g). The fillets were processed by one of the two methods, and the recovery of the marker in the suspensions was assessed by plate counting and qPCR targeting B. thermosphacta-rpoC. The same analyses were performed on six noninoculated fresh fillets. Stomaching and mechanical rinsing allowed efficient and repeatable recovery of the bacterial communities from the 42 inoculated fillets. No significant differences in recovery ratios were observed between the marker enumerated in the inoculation suspensions and in the corresponding recovery suspensions after rinsing and stomaching. However, the stomaching method allowed too many particles to pass through the filters bag, making necessary a limiting supplementary filtration step. As a consequence, only the rinsing recovery method allowed proper PCR quantification of the inoculated B. thermosphacta. The mean recovered bacterial level of the fillets was approximately 3 log CFU/g. It seems more relevant and cost-effective to recover the endogenous bacterial microbiota of a fish fillet structure using the rinsing method rather than the stomaching method.

International audience; Brochothrix thermosphacta is one of the main spoilers in food, responsible for meat and seafood spoilage through the production of malodorous volatile organic compounds. The molecules produced by this bacterium depend on the substrate (meat or seafood) and the storage conditions such as gas mixtures used in the packaging. It seems also that the spoilage potential is strain dependent as production of diacetyl and acetoin, two molecules responsible for seafood spoilage, varies with strains. Therefore, this suggests the involvement of different metabolic functions depending on both food substrate and strain capacities. In this study, we selected two strains with different abilities to produce diacetyl and acetoin and compared their behavior after grown in beef or cooked peeled shrimp juices. We determined the genes upregulated by both strains depending on the growth substrate and those that were specifically upregulated in only one strain. The genes upregulated by both strains in meat or in shrimp juice revealed the importance of the substrate for inducing specific metabolic pathways. The examination of genes that were specifically upregulated in only one of the two strains revealed strain features associated to specific substrates and also strain-specific regulations of metabolic pathways putatively leading to different levels of spoilage molecule production. This shows that the spoilage potential of B. thermosphacta depends on nutrients provided by food substrate and on metabolic activity potential that each strain possesses.

Brochothrix thermosphacta is one of the dominant bacterial species associated with spoilage of chilled meat and seafood products through the production of various metabolites responsible for off-odors. However, metabolic pathways leading to meat and seafood spoilage are not all well known. The production of spoiling molecules seems to depend both on strains and on food matrix. Several B. thermosphacta genome sequences have been reported, all issued from meat isolates. Here, we report four genome sequences, one complete and three as drafts. The four B. thermosphacta strains CD 337, TAP 175, BSAS1 3, and EBP 3070 were isolated from different ecological niches (seafood or meat products either spoiled or not and bovine slaughterhouse). These strains known as phenotypically and genetically different were selected to represent intraspecies diversity. CD 337 genome is 2,594,337 bp long, complete and circular, containing 2593 protein coding sequences and 28 RNA genes. TAP 175, BSAS1 3, and EBP 3070 genomes are arranged in 57, 83, and 71 contigs, containing 2515, 2668, and 2611 protein-coding sequences, respectively. These genomes were compared with two other B. thermosphacta complete genome sequences. The main genome content differences between strains are phages, plasmids, restriction/modification systems, and cell surface functions, suggesting a similar metabolic potential but a different niche adaptation capacity. Electronic supplementary material The online version of this article (10.1186/s40793-018-0333-z) contains supplementary material, which is available to authorized users.

International audience

Lactobacillus sakei is a lactic acid bacterium, naturally associated with long term storage of fresh meat at low temperature. Here we investigated the effect, on the evolution of pathogenic and spoilage microorganisms in ground beef, of L. sakei cocktails used as bioprotective cultures. We selectively developed a real time quantitative PCR method, allowing the quantification of individual L. sakei strains inoculated in ground meat with specific probes. Six cocktails of three strains were tested to evaluate their effect on the growth of Salmonella enterica Typhimurium, Escherichia coli O157:H7 and Brochothrix thermosphacta at 4°C and 8°C, under vacuum or modified atmosphere packaging. Using plating methods to quantify the different bacterial species, one cocktail showed an effect against S. Typhimurium and E. coli under given conditions. Real time quantitative PCR showed that the three inoculated L. sakei strains had a different growth pattern, and that the association of these three strains indeed impaired growth of S. Typhimurium and E. coli.

Brochothrix thermosphacta, a Cram-positive bacterium, is considered as the predominant spoilage microbiota of modified atmosphere packing (MAP) shrimp and fish. Traditional methods currently used to detect B. thermosphacta in foods are time-consuming and labour-intensive. The aim of this study was to develop a real-time PCR quantification method combined with a propidium monoazide (PMA) sample treatment step to monitor the population of B. thermosphacta in cooked shrimp and salmon. The specificity of the two primers M0405 and M0404 used to amplify a 70 bp fragment of the 16S rRNA gene was demonstrated by using purified DNA from 30 strains, among 21 bacterial species including 22 reference strains. Using these primers for real-time PCR and in pure culture, a good correlation was obtained between real-time PCR and the conventional plating method. Quantification was linear over 7-log units using artificially inoculated samples. The method performed successfully when tested on naturally contaminated cooked shrimp and fresh salmon, with a minimum threshold of 1.9 x 10(2) CFU/g for accurate quantification of B. the rmosphacta. The correlation between the B. thermosphacta counts obtained by real-time PCR and plate counts on naturally contaminated shrimp and salmon was high (R-2=0.895). Thus, this study presents a rapid tool for producing reliable quantitative data on B. thermosphacta in cooked shrimp and fresh salmon. (C) 2011 Elsevier Ltd. All rights reserved.

Aims: To investigate the antimicrobial spectrum of Lactococcus piscium CNCM I-4031 and its protective effect in cooked and peeled shrimp against Brochothrix thermosphacta. Methods and Results: Sixteen pathogenic and spoiling bacteria were inhibited in Elliker, but not in shrimp juice agar plates. In shrimp packed under modified atmosphere and stored at 8 degrees C, B. thermosphacta (103 CFU g-1) was inhibited by 4 center dot 1 log CFU g-1 when co-inoculated with L. piscium (106 CFU g-1). Brochothrix thermosphacta spoiled the product after 11 days, with the emission of strong butter/caramel off-odours. In co-culture with L. piscium, sensory shelf-life was extended by at least 10 days. The inhibition was partially explained by a drop in pH from 6 center dot 6 to 5 center dot 6. The physicochemical composition of shrimp and shrimp juice was established to identify the inhibition mechanisms involved. Conclusion: Lactococcus piscium CNCM I-4031 has a wide antimicrobial spectrum. The strain inhibits B. thermosphacta in shrimp and significantly prolongs sensory shelf-life. Significance and Impact of the Study: Lactococcus piscium CNCM I-4031 is shown to be a promising agent for improving shrimp quality and may be tested against pathogens and in other food matrices. Knowledge of the physicochemical composition of shrimp and shrimp juice will allow the development of a chemically defined model medium for determining the inhibition mechanisms involved.

In order to extend meat shelf-life, normal-pH and high-pH beef steaks were packaged under vacuum or in 100% C 0 2 atmosphere. Microbiological, colour (L*, a*, b*) and reflectance (R630-R580 values) characteristics were measured. After packaging under vacuum, Enterobacteriaceae, Brochothrix thermosphacta and Pseudomonas numbers were 10- to 100-fold greater on high-pH meat than on normal-pH meat. Packaging under C 0 2 improved the shelf-life of meat, particularly that of high-pH meat up to 42 days. For both high- and normal-pH meats, the bacterial flora was composed only of lactic acid bacteria. Normal-pH meat in C 0 2 atmosphere and vacuum packaging had a purple colour. After C02-pack opening the meat colour became bright red and R630- R580 measurements were high, whereas after opening of vacuum packaging R630-R580 decreased rapidly with increasing aerobic exposure. High-pH meat became paler with increasing storage time in C02. Simultaneously R630-R580 values increased and the pH fell. In addition it lost less exudate and oxidized less in air than the normal-pH meat under the same conditions.