Evolution of fungal community associated with ready-to-eat pineapple during storage under different temperature conditions.

The international market of fresh-cut products has witnessed dramatic growth in recent years, stimulated by consumer's demand for healthy, nutritious and convenient foods. One of the main challenging issues for the quality and safety of these products is the potential microbial spoilage that can significantly reduce their shelf-life. The complete identification of fresh-cut product microbiota together with the evaluation of environmental factors impact on microbial composition is of primary importance. We therefore assessed the fungal communities associated with the spoilage of ready-to-eat (RTE) pineapple using a metagenetic amplicon sequencing approach, based on the ITS2 region. Our results revealed a significant variability on fungal species composition between the different batches of RTE pineapple. The initial microbiota composition was the main influencing factor and determined the progress of spoilage. Temperature and storage time were the secondary factors influencing spoilage and their impact was depending on the initial prevalent fungal species, which showed different responses to the various modifications. Our results strongly suggest that further large-scale sampling of RTE pineapple production should be conducted in order to assess the full biodiversity range of fungal community involved in the spoilage process and for unravelling the impact of important environmental factors shaping the initial microbiota.

Heme Uptake in Lactobacillus sakei Evidenced by a New Energy Coupling Factor (ECF)-Like Transport System.

Lactobacillus sakei is a nonpathogenic lactic acid bacterium and a natural inhabitant of meat ecosystems. Although red meat is a heme-rich environment, L. sakei does not need iron or heme for growth, although it possesses a heme-dependent catalase. Iron incorporation into L. sakei from myoglobin and hemoglobin was previously shown by microscopy and the L. sakei genome reveals the complete equipment for iron and heme transport. Here, we report the characterization of a five-gene cluster (from lsa1836 to lsa1840 [lsa1836-1840]) encoding a putative metal iron ABC transporter. Interestingly, this cluster, together with a heme-dependent catalase gene, is also conserved in other species from the meat ecosystem. Our bioinformatic analyses revealed that the locus might correspond to a complete machinery of an energy coupling factor (ECF) transport system. We quantified in vitro the intracellular heme in the wild type (WT) and in our Δlsa1836-1840 deletion mutant using an intracellular heme sensor and inductively coupled plasma mass spectrometry for quantifying incorporated 57Fe heme. We showed that in the WT L. sakei, heme accumulation occurs rapidly and massively in the presence of hemin, while the deletion mutant was impaired in heme uptake; this ability was restored by in trans complementation. Our results establish the main role of the L. sakei Lsa1836-1840 ECF-like system in heme uptake. Therefore, this research outcome sheds new light on other possible functions of ECF-like systems.IMPORTANCELactobacillus sakei is a nonpathogenic bacterial species exhibiting high fitness in heme-rich environments such as meat products, although it does not need iron or heme for growth. Heme capture and utilization capacities are often associated with pathogenic species and are considered virulence-associated factors in the infected hosts. For these reasons, iron acquisition systems have been deeply studied in such species, while for nonpathogenic bacteria the information is scarce. Genomic data revealed that several putative iron transporters are present in the genome of the lactic acid bacterium L. sakei In this study, we demonstrate that one of them is an ECF-like ABC transporter with a functional role in heme transport. Such evidence has not yet been brought for an ECF; therefore, our study reveals a new class of heme transport system.

Large microbiota survey reveals how the microbial ecology of cooked ham is shaped by different processing steps.

Cooked ham production involves numerous steps shaping the microbial communities of the final product, with consequences on spoilage metabolites production. To identify the main factors driving the ecology of ham and its spoilage, we designed a study encompassing five variables related to ham production: type of storage during meat transportation, churning speed, drain-off time, slicing line and O2 packaging permeability. About 200 samples from the same facility were obtained and characterized with respect to i) their microbiota based on gyrB amplicon sequencing ii) their production of spoilage-related metabolites based on E-Nose analysis and enzymatic assays. The slicing was the most critical step, shaping two general types of microbiota according to the slicing line: one dominated by Carnobacterium divergens and another one dominated by Leuconostoc carnosum and Serratia proteamaculans. Regarding metabolites production, L. carnosum was associated to d-lactic acid, ethanol and acetic acid production, whereas Serratia proteamaculans was associated to acetic acid production. This last species prevailed with highly O2-permeable packaging. Within a given slicing line, campaign-based variations were observed, with Lactobacillus sakei, Leuconostoc mesenteroides and Carnobacterium maltaromaticum prevalent in summer. L. sakei was associated with l-lactic acid production and C. maltaromaticum with formic and acetic acid productions.

Viral metagenomic analysis of the cheese surface: A comparative study of rapid procedures for extracting viral particles.

The structure and functioning of microbial communities from fermented foods, including cheese, have been extensively studied during the past decade. However, there is still a lack of information about both the occurrence and the role of viruses in modulating the function of this type of spatially structured and solid ecosystems. Viral metagenomics was recently applied to a wide variety of environmental samples and standardized procedures for recovering viral particles from different type of materials has emerged. In this study, we adapted a procedure originally developed to extract viruses from fecal samples, in order to enable efficient virome analysis of cheese surface. We tested and validated the positive impact of both addition of a filtration step prior to virus concentration and substitution of purification by density gradient ultracentrifugation by a simple chloroform treatment to eliminate membrane vesicles. Viral DNA extracted from the several procedures, as well as a vesicle sample, were sequenced using Illumina paired-end MiSeq technology and the subsequent clusters assembled from the virome were analyzed to assess those belonging to putative phages, plasmid-derived DNA, or even from bacterial chromosomal DNA. The best procedure was then chosen, and used to describe the first cheese surface virome, using Epoisses cheese as example. This study provides the basis of future investigations regarding the ecological importance of viruses in cheese microbial ecosystems.

Reducing Salt in Raw Pork Sausages Increases Spoilage and Correlates with Reduced Bacterial Diversity.

UNLABELLED: Raw sausages are perishable foodstuffs; reducing their salt content raises questions about a possible increased spoilage of these products. In this study, we evaluated the influence of salt reduction (from 2.0% to 1.5% [wt/wt]), in combination with two types of packaging (modified atmosphere [50% mix of CO2-N2] and vacuum packaging), on the onset of spoilage and on the diversity of spoilage-associated bacteria. After 21 days of storage at 8°C, spoilage was easily observed, characterized by noticeable graying of the products and the production of gas and off-odors defined as rancid, sulfurous, or sour. At least one of these types of spoilage occurred in each sample, and the global spoilage intensity was more pronounced in samples stored under modified atmosphere than under vacuum packaging and in samples with the lower salt content. Metagenetic 16S rRNA pyrosequencing revealed that vacuum-packaged samples contained a higher total bacterial richness (n = 69 operational taxonomic units [OTUs]) than samples under the other packaging condition (n = 46 OTUs). The core community was composed of 6 OTUs (Lactobacillus sakei, Lactococcus piscium, Carnobacterium divergens, Carnobacterium maltaromaticum, Serratia proteamaculans, and Brochothrix thermosphacta), whereas 13 OTUs taxonomically assigned to the Enterobacteriaceae, Enterococcaceae, and Leuconostocaceae families comprised a less-abundant subpopulation. This subdominant community was significantly more abundant when 2.0% salt and vacuum packaging were used, and this correlated with a lower degree of spoilage. Our results demonstrate that salt reduction, particularly when it is combined with CO2-enriched packaging, promotes faster spoilage of raw sausages by lowering the overall bacterial diversity (both richness and evenness). IMPORTANCE: Our study takes place in the context of raw meat product manufacturing and is linked to a requirement for salt reduction. Health guidelines are calling for a reduction in dietary salt intake. However, salt has been used for a very long time as a hurdle technology, and salt reduction in meat products raises the question of spoilage and waste of food. The study was conceived to assess the role of sodium chloride reduction in meat products, both at the level of spoilage development and at the level of bacterial diversity, using 16S rRNA amplicon sequencing and raw pork sausage as a meat model.

High genetic diversity among strains of the unindustrialized lactic acid bacterium Carnobacterium maltaromaticum in dairy products as revealed by multilocus sequence typing.

Dairy products are colonized with three main classes of lactic acid bacteria (LAB): opportunistic bacteria, traditional starters, and industrial starters. Most of the population structure studies were previously performed with LAB species belonging to these three classes and give interesting knowledge about the population structure of LAB at the stage where they are already industrialized. However, these studies give little information about the population structure of LAB prior their use as an industrial starter. Carnobacterium maltaromaticum is a LAB colonizing diverse environments, including dairy products. Since this bacterium was discovered relatively recently, it is not yet commercialized as an industrial starter, which makes C. maltaromaticum an interesting model for the study of unindustrialized LAB population structure in dairy products. A multilocus sequence typing scheme based on an analysis of fragments of the genes dapE, ddlA, glpQ, ilvE, pyc, pyrE, and leuS was applied to a collection of 47 strains, including 28 strains isolated from dairy products. The scheme allowed detecting 36 sequence types with a discriminatory index of 0.98. The whole population was clustered in four deeply branched lineages, in which the dairy strains were spread. Moreover, the dairy strains could exhibit a high diversity within these lineages, leading to an overall dairy population with a diversity level as high as that of the nondairy population. These results are in agreement with the hypothesis according to which the industrialization of LAB leads to a diversity reduction in dairy products.

Integration of metataxonomic data sets into microbial association networks highlights shared bacterial community dynamics in fermented vegetables.

The management of food fermentation is still largely based on empirical knowledge, as the dynamics of microbial communities and the underlying metabolic networks that produce safe and nutritious products remain beyond our understanding. Although these closed ecosystems contain relatively few taxa, they have not yet been thoroughly characterized with respect to how their microbial communities interact and dynamically evolve. However, with the increased availability of metataxonomic data sets on different fermented vegetables, it is now possible to gain a comprehensive understanding of the microbial relationships that structure plant fermentation. In this study, we applied a network-based approach to the integration of public metataxonomic 16S data sets targeting different fermented vegetables throughout time. Specifically, we aimed to explore, compare, and combine public 16S data sets to identify shared associations between amplicon sequence variants (ASVs) obtained from independent studies. The workflow includes steps for searching and selecting public time-series data sets and constructing association networks of ASVs based on co-abundance metrics. Networks for individual data sets are then integrated into a core network, highlighting significant associations. Microbial communities are identified based on the comparison and clustering of ASV networks using the "stochastic block model" method. When we applied this method to 10 public data sets (including a total of 931 samples) targeting five varieties of vegetables with different sampling times, we found that it was able to shed light on the dynamics of vegetable fermentation by characterizing the processes of community succession among different bacterial assemblages. IMPORTANCE: Within the growing body of research on the bacterial communities involved in the fermentation of vegetables, there is particular interest in discovering the species or consortia that drive different fermentation steps. This integrative analysis demonstrates that the reuse and integration of public microbiome data sets can provide new insights into a little-known biotope. Our most important finding is the recurrent but transient appearance, at the beginning of vegetable fermentation, of amplicon sequence variants (ASVs) belonging to Enterobacterales and their associations with ASVs belonging to Lactobacillales. These findings could be applied to the design of new fermented products.

Catabolism of N-acetylneuraminic acid, a fitness function of the food-borne lactic acid bacterium Lactobacillus sakei, involves two newly characterized proteins.

In silico analysis of the genome sequence of the meat-borne lactic acid bacterium (LAB) Lactobacillus sakei 23K has revealed a repertoire of potential functions related to the adaptation of this bacterium to the meat environment. Among these functions, the ability to use N-acetyl-neuraminic acid (NANA) as a carbon source could provide a competitive advantage for growth on meat in which this amino sugar is present. In this work, we proposed to analyze the functionality of a gene cluster encompassing nanTEAR and nanK (nanTEAR-nanK). We established that this cluster encoded a pathway allowing transport and early steps of the catabolism of NANA in this genome. We also demonstrated that this cluster was absent from the genome of other L. sakei strains that were shown to be unable to grow on NANA. Moreover, L. sakei 23K nanA, nanT, nanK, and nanE genes were able to complement Escherichia coli mutants. Construction of different mutants in L. sakei 23K ΔnanR, ΔnanT, and ΔnanK and the double mutant L. sakei 23K Δ(nanA-nanE) made it possible to show that all were impaired for growth on NANA. In addition, two genes located downstream from nanK, lsa1644 and lsa1645, are involved in the catabolism of sialic acid in L. sakei 23K, as a L. sakei 23K Δlsa1645 mutant was no longer able to grow on NANA. All these results demonstrate that the gene cluster nanTEAR-nanK-lsa1644-lsa1645 is indeed involved in the use of NANA as an energy source by L. sakei.

Holistic integration of omics data reveals the drivers that shape the ecology of microbial meat spoilage scenarios.

Background: The use of omics data for monitoring the microbial flow of fresh meat products along a production line and the development of spoilage prediction tools from these data is a promising but challenging task. In this context, we produced a large multivariate dataset (over 600 samples) obtained on the production lines of two similar types of fresh meat products (poultry and raw pork sausages). We describe a full analysis of this dataset in order to decipher how the spoilage microbial ecology of these two similar products may be shaped differently depending on production parameter characteristics. Methods: Our strategy involved a holistic approach to integrate unsupervised and supervised statistical methods on multivariate data (OTU-based microbial diversity; metabolomic data of volatile organic compounds; sensory measurements; growth parameters), and a specific selection of potential uncontrolled (initial microbiota composition) or controlled (packaging type; lactate concentration) drivers. Results: Our results demonstrate that the initial microbiota, which is shown to be very different between poultry and pork sausages, has a major impact on the spoilage scenarios and on the effect that a downstream parameter such as packaging type has on the overall evolution of the microbial community. Depending on the process, we also show that specific actions on the pork meat (such as deboning and defatting) elicit specific food spoilers such as Dellaglioa algida, which becomes dominant during storage. Finally, ecological network reconstruction allowed us to map six different metabolic pathways involved in the production of volatile organic compounds involved in spoilage. We were able connect them to the different bacterial actors and to the influence of packaging type in an overall view. For instance, our results demonstrate a new role of Vibrionaceae in isopropanol production, and of Latilactobacillus fuchuensis and Lactococcus piscium in methanethiol/disylphide production. We also highlight a possible commensal behavior between Leuconostoc carnosum and Latilactobacillus curvatus around 2,3-butanediol metabolism. Conclusion: We conclude that our holistic approach combined with large-scale multi-omic data was a powerful strategy to prioritize the role of production parameters, already known in the literature, that shape the evolution and/or the implementation of different meat spoilage scenarios.

Fingerprint of lactic acid bacteria population in beef carpaccio is influenced by storage process and seasonal changes.

We have investigated the population structure of lactic acid bacteria (LAB) for several beef carpaccio available on the market with the purpose of comparing the effect of storage process (modified-atmosphere packaging and vacuum-packaging) and of seasonal changes on this microbial population. Out of 60 samples we have characterised 214 isolates accounting for 10 LAB species and 35 isolates accounting for 11 non-LAB species. Lactobacillus sakei, Leuconostoc carnosum and Leuconostoc mesenteroides were the most prevailing LAB species with a frequency of identification within 66%, 62% and 52% of the samples respectively. These 3 species were also characterised by a phenotypic intra-species diversity of isolates based on colony morphology. We showed that the prevalence was increased 1.5 fold for L. sakei and L. mesenteroides during the summer sampling in comparison to the spring or the fall sampling suggesting an environmental origin of these two species. Seasonal variations were also observed for the prevalence of Lactobacillus fuchuensis and L. carnosum in spring (2- and 1.5-fold increase, respectively) and of Brochothrix thermosphacta in fall (6-fold increase). Finally, we demonstrated that the growth potential after the sell-by-date was favourable of 1.25 log(10) cfu g(-1) to Leuconostoc spp. in modified-atmosphere packaging and of 1.38 log(10) cfu g(-1) to Lactobacillus spp. in vacuum-packaging. In conclusion, we show that important and unsuspected traits in bacterial population dynamics can be unravelled by large sampling strategies. We discuss about the need to take this assessment into account for further studies on bacterial ecosystems of meat.

Intraspecies diversity of Lactobacillus sakei response to oxidative stress and variability of strain performance in mixed strains challenges.

Lactobacillus sakei is a meat-borne lactic acid bacterium species exhibiting a wide genomic diversity. We have investigated the diversity of response to various oxidative compounds, between L. sakei strains, among a collection representing the genomic diversity. We observed various responses to the different compounds as well as a diversity of response depending on the aeration conditions used for cell growth. A principal component analysis revealed two main phenotypic groups, partially correlating with previously described genomic clusters. We designed strains mixes composed of three different strains, in order to examine the behavior of each strain, when cultured alone or in the presence of other strains. The strains composing the mixtures were chosen as diverse as possible, i.e. exhibiting diverse responses to oxidative stress and belonging to different genomic clusters. Growth and survival rates of each strain were monitored under various aeration conditions, with or without heme supplementation. The results obtained suggest that some strains may act as "helper" or "burden" strains depending on the oxidative conditions encountered during incubation. This study confirms that resistance to oxidative stress is extremely variable within the L. sakei species and that this property should be considered when investigating starter performance in the complex meat bacterial ecosystems.

Metagenetic characterization of bacterial communities associated with ready-to-eat leafy vegetables and study of temperature effect on their composition during storage.

Ready-to-eat (RTE) and fresh-cut vegetables meet the current needs for healthy and easy-to-prepare food. However, raw vegetables are widely known to harbor large and diverse bacterial communities promoting spoilage and reducing their shelf-life. A better understanding of their bacterial community and the impact of various environmental factors on its composition is essential to ensure the production of high-quality fresh-cut produce. Therefore, a metagenetic amplicon approach, based on gyrB sequencing, was applied for deciphering the bacterial communities associated with the spoilage of RTE rocket and baby spinach and monitoring the changes occurring in their composition during storage at different temperatures. Our results indicated that Pseudomonas genus was the main spoilage group for both leafy vegetables. Specifically, Pseudomonas viridiflava was dominant in most samples of rocket, while a new Pseudomonas species as well as, Pseudomonas fluorescens and/or Pseudomonas fragi were highly abundant in baby spinach. A significant variability on bacterial species composition among different batches of each vegetable type was observed. In the case of baby spinach, the impact of temperature and/or storage time on bacterial microbiota was not explicitly revealed at batch-level. Concerning rocket, the storage time was the most influential factor resulting in the reduction of Pseudomonas species' abundances and the parallel increase of lactic acid bacteria abundances. The results suggest that a large-scale sampling and further investigation of the various environmental factors shaping the microbiota are needed for gaining deeper knowledge of the diverse bacterial communities on RTE leafy vegetables and thus, enhance the quality of these products.

Combination of High-Pressure Treatment at 500 MPa and Biopreservation with a Lactococcus lactis Strain for Lowering the Bacterial Growth during Storage of Diced Cooked Ham with Reduced Nitrite Salt.

We investigated the combined effects of biopreservation and high-pressure treatment on bacterial communities of diced cooked ham prepared with diminished nitrite salt. First, bacterial communities of four commercial brands of diced cooked ham from local supermarkets were characterized and stored frozen. Second, sterile diced cooked ham, prepared with reduced levels of nitrite, was inoculated with two different microbiota collected from the aforementioned commercial samples together with a nisin-producing Lactococcus lactis protective strain able to recover from a 500 MPa high-pressure treatment. Samples were then treated at 500 MPa for 5 min, and bacterial dynamics were monitored during storage at 8 °C. Depending on samples, the ham microbiota was dominated by different Proteobacteria (Pseudomonas, Serratia, Psychrobacter, or Vibrio) or by Firmicutes (Latilactobacillus and Leuconostoc). Applied alone, none of the treatments stabilized during the growth of the ham microbiota. Nevertheless, the combination of biopreservation and high-pressure treatment was efficient in reducing the growth of Proteobacteria spoilage species. However, this effect was dependent on the nature of the initial microbiota, showing that the use of biopreservation and high-pressure treatment, as an alternative to nitrite reduction for ensuring cooked ham microbial safety, merits attention but still requires improvement.

Application of a path-modelling approach for deciphering causality relationships between microbiota, volatile organic compounds and off-odour profiles during meat spoilage.

Microbiological spoilage of meat is considered as a process which involves mainly bacterial metabolism leading to degradation of meat sensory qualities. Studying spoilage requires the collection of different types of experimental data encompassing microbiological, physicochemical and sensorial measurements. Within this framework, the objective herein was to carry out a multiblock path modelling workflow to decipher causality relationships between different types of spoilage-related responses: composition of microbiota, volatilome and off-odour profiles. Analyses were performed with the Path-ComDim approach on a large-scale dataset collected on fresh turkey sausages. This approach enabled to quantify the importance of causality relationships determined a priori between each type of responses as well as to identify important responses involved in spoilage, then to validate causality assumptions. Results were very promising: the data integration confirmed and quantified the causality between data blocks, exhibiting the dynamical nature of spoilage, mainly characterized by the evolution of off-odour profiles caused by the production of volatile organic compounds such as ethanol or ethyl acetate. This production was possibly associated with several bacterial species like Lactococcus piscium, Leuconostoc gelidum, Psychrobacter sp. or Latilactobacillus fuchuensis. Likewise, the production of acetoin and diacetyl in meat spoilage was highlighted. The Path-ComDim approach illustrated here with meat spoilage can be applied to other large-scale and heterogeneous datasets associated with pathway scenarios and represents a promising key tool for deciphering causality in complex biological phenomena.

Nucleotide sequence and analysis of pRC12 and pRC18, two theta-replicating plasmids harbored by Lactobacillus curvatus CRL 705.

The nucleotide sequences of plasmids pRC12 (12,342 bp; GC 43.99%) and pRC18 (18,664 bp; GC 34.33%), harbored by the bacteriocin-producer Lactobacillus curvatus CRL 705, were determined and analyzed. Plasmids pRC12 and pRC18 share a region with high DNA identity (> 83% identity between RepA, a Type II toxin-antitoxin system and a tyrosine integrase genes) and are stably maintained in their natural host L. curvatus CRL 705. Both plasmids are low copy number and belong to the theta-type replicating group. While pRC12 is a pUCL287-like plasmid that possesses iterons and the repA and repB genes for replication, pRC18 harbors a 168 amino acid replication protein affiliated to RepB, which was named RepB'. Plasmid pRC18 also possesses a pUCL287-like repA gene but it was disrupted by an 11 kb insertion element that contains RepB', several transposases/IS elements, and the lactocin Lac705 operon. An Escherichia coli / Lactobacillus shuttle vector, named plasmid p3B1, carrying the pRC18 replicon (i.e. repB' and replication origin), a chloramphenicol resistance gene and a pBluescript backbone, was constructed and used to define the host range of RepB'. Chloramphenicol-resistant transformants were obtained after electroporation of Lactobacillus plantarum CRL 691, Lactobacillus sakei 23K and a plasmid-cured derivative of L. curvatus CRL 705, but not of L. curvatus DSM 20019 or Lactococcus lactis NZ9000. Depending on the host, transformation efficiency ranged from 102 to 107 per µg of DNA; in the new hosts, the plasmid was relatively stable as 29-53% of recombinants kept it after cell growth for 100 generations in the absence of selective pressure. Plasmid p3B1 could therefore be used for cloning and functional studies in several Lactobacillus species.

Large-scale multivariate dataset on the characterization of microbiota diversity, microbial growth dynamics, metabolic spoilage volatilome and sensorial profiles of two industrially produced meat products subjected to changes in lactate concentration and packaging atmosphere.

Data in this article provide detailed information on the diversity of bacterial communities present on 576 samples of raw pork or poultry sausages produced industrially in 2017. Bacterial growth dynamics and diversity were monitored throughout the refrigerated storage period to estimate the impact of packaging atmosphere and the use of potassium lactate as chemical preservative. The data include several types of analysis aiming at providing a comprehensive microbial ecology of spoilage during storage and how the process parameters do influence this phenomenon. The analysis includes: the gas content in packaging, pH, chromametric measurements, plate counts (total mesophilic aerobic flora and lactic acid bacteria), sensorial properties of the products, meta-metabolomic quantification of volatile organic compounds and bacterial community metagenetic analysis. Bacterial diversity was monitored using two types of amplicon sequencing (16S rRNA and GyrB encoding genes) at different time points for the different conditions (576 samples for gyrB and 436 samples for 16S rDNA). Sequencing data were generated by using Illumina MiSeq. The sequencing data have been deposited in the bioproject PRJNA522361. Samples accession numbers vary from SAMN10964863 to SAMN10965438 for gyrB amplicon and from SAMN10970131 to SAMN10970566 for 16S.

Behavior of the meat-borne bacterium Lactobacillus sakei during its transit through the gastrointestinal tracts of axenic and conventional mice.

A Lactobacillus sakei strain named FLEC01 was isolated from human feces and characterized genotypically. Comparison of the genetic features of this strain with those of both the meat-borne L. sakei strain 23K and another human isolate, LTH5590, showed that they belong to different but closely related clusters. The three L. sakei strains did not persist and only transited through the gastrointestinal tracts (GITs) of conventional C3H/HeN mice. In contrast, they all colonized the GITs of axenic mice and rapidly reached a population of 10(9) CFU/g of feces, which remained stable until day 51. Five days after mice were fed, a first subpopulation, characterized by small colonies, appeared and reached 50% of the total L. sakei population in mice. Fifteen to 21 days after feeding, a second subpopulation, characterized by rough colonies, appeared. It coexisted with the two other populations until day 51, and its cell shapes were also affected, suggesting a dysfunction of the cell division or cell wall. No clear difference between the behaviors of the meat-borne strain and the two human isolates in both conventional and axenic mice was observed, suggesting that L. sakei is a food-borne bacterium rather than a commensal one and that its presence in human feces originates from diet. Previous observations of Escherichia coli strains suggest that the mouse GIT environment could induce mutations to increase their survival and colonization capacities. Here, we observed similar mutations concerning a food-grade gram-positive bacterium for the first time.

Intraspecies genomic diversity and natural population structure of the meat-borne lactic acid bacterium Lactobacillus sakei.

Lactobacillus sakei is a food-borne bacterium naturally found in meat and fish products. A study was performed to examine the intraspecies diversity among 73 isolates sourced from laboratory collections in several different countries. Pulsed-field gel electrophoresis analysis demonstrated a 25% variation in genome size between isolates, ranging from 1,815 kb to 2,310 kb. The relatedness between isolates was then determined using a PCR-based method that detects the possession of 60 chromosomal genes belonging to the flexible gene pool. Ten different strain clusters were identified that had noticeable differences in their average genome size reflecting the natural population structure. The results show that many different genotypes may be isolated from similar types of meat products, suggesting a complex ecological habitat in which intraspecies diversity may be required for successful adaptation. Finally, proteomic analysis revealed a slight difference between the migration patterns of highly abundant GapA isoforms of the two prevailing L. sakei subspecies (sakei and carnosus). This analysis was used to affiliate the genotypic clusters with the corresponding subspecies. These findings reveal for the first time the extent of intraspecies genomic diversity in L. sakei. Consequently, identification of molecular subtypes may in the future prove valuable for a better understanding of microbial ecosystems in food products.

The complete genome sequence of the meat-borne lactic acid bacterium Lactobacillus sakei 23K.

Lactobacillus sakei is a psychotrophic lactic acid bacterium found naturally on fresh meat and fish. This microorganism is widely used in the manufacture of fermented meats and has biotechnological potential in biopreservation and food safety. We have explored the 1,884,661-base-pair (bp) circular chromosome of strain 23K encoding 1,883 predicted genes. Genome sequencing revealed a specialized metabolic repertoire, including purine nucleoside scavenging that may contribute to an ability to successfully compete on raw meat products. Many genes appear responsible for robustness during the rigors of food processing--particularly resilience against changing redox and oxygen levels. Genes potentially responsible for biofilm formation and cellular aggregation that may assist the organism to colonize meat surfaces were also identified. This genome project is an initial step for investigating new biotechnological approaches to meat and fish processing and for exploring fundamental aspects of bacterial adaptation to these specific environments.

Draft Genome Sequences of Nine Strains of Brochothrix thermosphacta, Carnobacterium divergens, Lactobacillus algidus, Lactobacillus fuchuensis, Lactococcus piscium, Leuconostoc gelidum subsp. gasicomitatum, Pseudomonas lundensis, and Weissella viridescens, a Collection of Psychrotrophic Species Involved in Meat and Seafood Spoilage.

In this study, we present the draft genome sequences of nine strains from various psychrotrophic species identified in meat products and being recognized as important emerging food spoilers. Many of these species have only one or few strains being sequenced, and this work will contribute to the improvement of the overall genomic knowledge about them.