Genotypic and phenotypic characterization of the food spoilage bacterium Brochothrix thermosphacta.

Microbial food spoilage is responsible for significant economic losses. Brochothrix thermosphacta is one of the major bacteria involved in the spoilage of meat and seafood. Its growth and metabolic activities during food storage result in the production of metabolites associated with off-odors. In this study, we evaluated the genotypic and phenotypic diversity of this species. A collection of 161 B. thermosphacta strains isolated from different foods, spoiled or not, and from a slaughterhouse environment was constituted from various laboratory collections and completed with new isolates. A PCR test based on the rpoB gene was developed for a fast screening of B. thermosphacta isolates. Strains were typed by MALDI-TOF MS, rep-PCR, and PFGE. Each typing method separated strains into distinct groups, revealing significant intra-species diversity. These classifications did not correlate with the ecological origin of strains. The ability to produce acetoin and diacetyl, two molecules associated with B. thermosphacta spoilage, was evaluated in meat and shrimp juices. The production level was variable between strains and the spoilage ability on meat or shrimp juice did not correlate with the substrate origin of strains. Although the B. thermosphacta species encompasses ubiquitous strains, spoiling ability is both strain- and environment-dependent.

Diversity of bacterial communities in French chicken cuts stored under modified atmosphere packaging.

Poultry meat, the second most consumed meat in France, is commercialized mainly as portions of chicken cuts with various quality labels, stored under various modified atmosphere packaging (MAP), with shelf-life ranging from 9 to 17 days. We used 16S rDNA pyrosequencing to describe microbiota of chicken legs. Ten samples representing a wide diversity of labels and MAP available on the market were collected from local supermarkets and stored at 4 °C. Microbiota were collected, total DNA was extracted, and V1-V3 fragment of 16S rRNA genes were amplified and sequenced. For data analysis several pipelines were compared. The Qiime pipeline was chosen to cluster reads and we used a database previously developed for a meat and fish microbial ecology study. Variability between samples was observed and a listing of bacteria present on chicken meat was established. The structure of the bacterial communities were compared with traditional cultural methods and validated with quantitative real time PCR. Brochothrix thermosphacta, Pseudomonas sp., and Carnobacterium sp. were dominant and the nature of the gas used for packaging influenced the relative abundance of each suggesting a MAP gas composition dependent competition between these species. We also noticed that slaughterhouse environment may influence the nature of the contaminants.

Genes associated to lactose metabolism illustrate the high diversity of Carnobacterium maltaromaticum.

The dairy population of Carnobacterium maltaromaticum is characterized by a high diversity suggesting a high diversity of the genetic traits linked to the dairy process. As lactose is the main carbon source in milk, the genetics of lactose metabolism was investigated in this LAB. Comparative genomic analysis revealed that the species C. maltaromaticum exhibits genes related to the Leloir and the tagatose-6-phosphate (Tagatose-6P) pathways. More precisely, strains can bear genes related to one or both pathways and several strains apparently do not contain homologs related to these pathways. Analysis at the population scale revealed that the Tagatose-6P and the Leloir encoding genes are disseminated in multiple phylogenetic lineages of C. maltaromaticum: genes of the Tagatose-6P pathway are present in the lineages I, II and III, and genes of the Leloir pathway are present in the lineages I, III and IV. These data suggest that these genes evolved thanks to horizontal transfer, genetic duplication and translocation. We hypothesize that the lac and gal genes evolved in C. maltaromaticum according to a complex scenario that mirrors the high population diversity.

Influence of measurement and control of microaerobic gaseous atmospheres in methods for Campylobacter growth studies.

Campylobacter is the leading cause of bacterial enteritis in the world. For this reason, this pathogen is widely studied. As a microaerophilic and capnophilic microorganism, this foodborne pathogen requires an atmosphere with reduced oxygen (O2) and elevated carbon dioxide (CO2) concentrations for its optimal growth in vitro. According to the procedure for Campylobacter spp. isolation and cultivation from food products and environmental samples, European and American standards recommend gas proportions of 5% O2 and 10% CO2, complemented with nitrogen (N2). However, in the literature, the reported proportion of O2 for microaerobic growth conditions of Campylobacter spp. can range from 2.5% to 15% and the reason for this variation is usually not explained. The use of different gas generating systems and media to detect and to grow Campylobacter from foodstuff and the lack of information about gas producing systems are the main sources of the loss of consistancy between data. In this review, the relevance, strengths and weaknesses of these methods and their impact on Campylobacter biology are discussed. In conclusion the minimum information concerning microaerobic gaseous atmospheres are suggested in order to better harmonize data obtained from research studies for a better understanding of Campylobacter features.

Bacterial spoilers of food: behavior, fitness and functional properties.

Most food products are highly perishable as they constitute a rich nutrient source for microbial development. Among the microorganisms contaminating food, some present metabolic activities leading to spoilage. In addition to hygienic rules to reduce contamination, various treatments are applied during production and storage to avoid the growth of unwanted microbes. The nature and appearance of spoilage therefore depend on the physiological state of spoilers and on their ability to resist the processing/storage conditions and flourish on the food matrix. Spoilage also relies on the interactions between the microorganisms composing the ecosystems encountered in food. The recent rapid increase in publicly available bacterial genome sequences, as well as the access to high-throughput methods, should lead to a better understanding of spoiler behavior and to the possibility of decreasing food spoilage. This review lists the main bacterial species identified as food spoilers, their ability to develop during storage and/or processing, and the functions potentially involved in spoilage. We have also compiled an inventory of the available genome sequences of species encompassing spoilage strains. Combining in silico analysis of genome sequences with experimental data is proposed in order to understand and thus control the bacterial spoilage of food better.

Transcriptomic and proteomic analysis of Oenococcus oeni PSU-1 response to ethanol shock.

The correct development of malolactic fermentation depends on the capacity of Oenococcus oeni to survive under harsh wine conditions. The presence of ethanol is one of the most stressful factors affecting O. oeni performance. In this study, the effect of ethanol addition (12% vol/vol) on O. oeni PSU-1 has been evaluated using a transcriptomic and proteomic approach. Transcriptomic analysis revealed that the main functional categories of the genes affected by ethanol were metabolite transport and cell wall and membrane biogenesis. It was also observed that some genes were over-expressed in response to ethanol stress (for example, the heat shock protein Hsp20 and a dipeptidase). Proteomic analysis showed that several proteins are affected by the presence of ethanol. Functions related to protein synthesis and stability are the main target of ethanol damage. In some cases the decrease in protein concentration could be due to the relocation of cytosolic proteins in the membrane, as a protective mechanism. The omic approach used to study the response of O. oeni to ethanol highlights the importance of the cell membrane in the global stress response and opens the door to future studies on this issue.

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.

A Bayesian Approach to Describe and Simulate the pH Evolution of Fresh Meat Products Depending on the Preservation Conditions.

Measuring the pH of meat products during storage represents an efficient way to monitor microbial spoilage, since pH is often linked to the growth of several spoilage-associated microorganisms under different conditions. The present work aimed to develop a modelling approach to describe and simulate the pH evolution of fresh meat products, depending on the preservation conditions. The measurement of pH on fresh poultry sausages, made with several lactate formulations and packed under three modified atmospheres (MAP), from several industrial production batches, was used as case-study. A hierarchical Bayesian approach was developed to better adjust kinetic models while handling a low number of measurement points. The pH changes were described as a two-phase evolution, with a first decreasing phase followed by a stabilisation phase. This stabilisation likely took place around the 13th day of storage, under all the considered lactate and MAP conditions. The effects of lactate and MAP on pH previously observed were confirmed herein: (i) lactate addition notably slowed down acidification, regardless of the packaging, whereas (ii) the 50%CO2-50%N2 MAP accelerated the acidification phase. The Bayesian modelling workflow-and the script-could be used for further model adaptation for the pH of other food products and/or other preservation strategies.

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.

Contribution of omics to biopreservation: Toward food microbiome engineering.

Biopreservation is a sustainable approach to improve food safety and maintain or extend food shelf life by using beneficial microorganisms or their metabolites. Over the past 20 years, omics techniques have revolutionised food microbiology including biopreservation. A range of methods including genomics, transcriptomics, proteomics, metabolomics and meta-omics derivatives have highlighted the potential of biopreservation to improve the microbial safety of various foods. This review shows how these approaches have contributed to the selection of biopreservation agents, to a better understanding of the mechanisms of action and of their efficiency and impact within the food ecosystem. It also presents the potential of combining omics with complementary approaches to take into account better the complexity of food microbiomes at multiple scales, from the cell to the community levels, and their spatial, physicochemical and microbiological heterogeneity. The latest advances in biopreservation through omics have emphasised the importance of considering food as a complex and dynamic microbiome that requires integrated engineering strategies to increase the rate of innovation production in order to meet the safety, environmental and economic challenges of the agri-food sector.

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.

Complete genome sequence of the probiotic Lactobacillus casei strain BL23.

The entire genome of Lactobacillus casei BL23, a strain with probiotic properties, has been sequenced. The genomes of BL23 and the industrially used probiotic strain Shirota YIT 9029 (Yakult) seem to be very similar.

Analysis of Lactobacillus sakei mutants selected after adaptation to the gastrointestinal tracts of axenic mice.

We recently showed that Lactobacillus sakei, a natural meat-borne lactic acid bacterium, can colonize the gastrointestinal tracts (GIT) of axenic mice but that this colonization in the intestinal environment selects L. sakei mutants showing modified colony morphology (small and rough) and cell shape, most probably resulting from the accumulation of various mutations that confer a selective advantage for persistence in the GIT. In the present study, we analyzed such clones, issued from three different L. sakei strains, in order to determine which functions were modified in the mutants. In the elongated filamentous cells of the rough clones, transmission electron microscopy (TEM) analysis showed a septation defect and dotted and slanted black bands, suggesting the presence of a helical structure around the cells. Comparison of the cytoplasmic and cell wall/membrane proteomes of the meat isolate L. sakei 23K and of one of its rough derivatives revealed a modified expression for 38 spots. The expression of six oxidoreductases, several stress proteins, and four ABC transporters was strongly reduced in the GIT-adapted strain, while the actin-like MreB protein responsible for cell shaping was upregulated. In addition, the expression of several enzymes involved in carbohydrate metabolism was modified, which may correlate with the observation of modified growth of mutants on various carbon sources. These results suggest that the modifications leading to a better adaptation to the GIT are pleiotropic and are characterized in a rough mutant by a different stress status, a cell wall modification, and modified use of energy sources, leading to an improved fitness for the colonization of the GIT.

Iron sources used by the nonpathogenic lactic acid bacterium Lactobacillus sakei as revealed by electron energy loss spectroscopy and secondary-ion mass spectrometry.

Lactobacillus sakei is a lactic acid bacterium naturally found on meat. Although it is generally acknowledged that lactic acid bacteria are rare species in the microbial world which do not have iron requirements, the genome sequence of L. sakei 23K has revealed quite complete genetic equipment dedicated to transport and use of this metal. Here, we aimed to investigate which iron sources could be used by this species as well as their role in the bacterium's physiology. Therefore, we developed a microscopy approach based on electron energy loss spectroscopy (EELS) analysis and nano-scale secondary-ion mass spectrometry (SIMS) in order to analyze the iron content of L. sakei cells. This revealed that L. sakei can use iron sources found in its natural ecosystem, myoglobin, hemoglobin, hematin, and transferrin, to ensure long-term survival during stationary phase. This study reveals that analytical image methods (EELS and SIMS) are powerful complementary tools for investigation of metal utilization by bacteria.

Proteomic comparison of the cytosolic proteins of three Bifidobacterium longum human isolates and B. longum NCC2705.

BACKGROUND: Bifidobacteria are natural inhabitants of the human gastrointestinal tract. In full-term newborns, these bacteria are acquired from the mother during delivery and rapidly become the predominant organisms in the intestinal microbiota. Bifidobacteria contribute to the establishment of healthy intestinal ecology and can confer health benefits to their host. Consequently, there is growing interest in bifidobacteria, and various strains are currently used as probiotic components in functional food products. However, the probiotic effects have been reported to be strain-specific. There is thus a need to better understand the determinants of the observed benefits provided by these probiotics. Our objective was to compare three human B. longum isolates with the sequenced model strain B. longum NCC2705 at the chromosome and proteome levels. RESULTS: Pulsed field electrophoresis genotyping revealed genetic heterogeneity with low intraspecies strain relatedness among the four strains tested. Using two-dimensional gel electrophoresis, we analyzed qualitative differences in the cytosolic protein patterns. There were 45 spots that were present in some strains and absent in others. Spots were excised from the gels and subjected to peptide mass fingerprint analysis for identification. The 45 spots represented 37 proteins, most of which were involved in carbohydrate metabolism and cell wall or cell membrane synthesis. Notably, the protein patterns were correlated with differences in cell membrane properties like surface hydrophobicity and cell agglutination. CONCLUSION: These results showed that proteomic analysis can be valuable for investigating differences in bifidobacterial species and may provide a better understanding of the diversity of bifidobacteria and their potential use as probiotics.

Primary metabolism in Lactobacillus sakei food isolates by proteomic analysis.

BACKGROUND: Lactobacillus sakei is an important food-associated lactic acid bacterium commonly used as starter culture for industrial meat fermentation, and with great potential as a biopreservative in meat and fish products. Understanding the metabolic mechanisms underlying the growth performance of a strain to be used for food fermentations is important for obtaining high-quality and safe products. Proteomic analysis was used to study the primary metabolism in ten food isolates after growth on glucose and ribose, the main sugars available for L. sakei in meat and fish. RESULTS: Proteins, the expression of which varied depending on the carbon source were identified, such as a ribokinase and a D-ribose pyranase directly involved in ribose catabolism, and enzymes involved in the phosphoketolase and glycolytic pathways. Expression of enzymes involved in pyruvate and glycerol/glycerolipid metabolism were also affected by the change of carbon source. Interestingly, a commercial starter culture and a protective culture strain down-regulated the glycolytic pathway more efficiently than the rest of the strains when grown on ribose. The overall two-dimensional gel electrophoresis (2-DE) protein expression pattern was similar for the different strains, though distinct differences were seen between the two subspecies (sakei and carnosus), and a variation of about 20% in the number of spots in the 2-DE gels was observed between strains. A strain isolated from fermented fish showed a higher expression of stress related proteins growing on both carbon sources. CONCLUSIONS: It is obvious from the data obtained in this study that the proteomic approach efficiently identifies differentially expressed proteins caused by the change of carbon source. Despite the basic similarity in the strains metabolic routes when they ferment glucose and ribose, there were also interesting differences. From the application point of view, an understanding of regulatory mechanisms, actions of catabolic enzymes and proteins, and preference of carbon source is of great importance.

Spoilage of Chilled Fresh Meat Products during Storage: A Quantitative Analysis of Literature Data.

A literature search was performed on spoilage of fresh meat products by combining keyword query, text mining and expert elicitation. From the 258 collected studies, a quantitative analysis was first performed to identify the methods which are the most used to evaluate spoilage beside the preservation strategies suggested. In a second step focusing on a subset of 24 publications providing quantitative data on spoilage occurrence time, associations between spoilage occurrence time of meat products and specific spoilage indicators were investigated. The analysis especially focused on factors well represented in the 24 publications, i.e., gas packaging (O2 and CO2) and storage temperature. Relationships between spoilage occurrence and several microbiological indicators were also sought. The results point out possible advantages of removing dioxygen in packaging to delay spoilage occurrence, whereas, in the presence of dioxygen, the carbon dioxide proportion in the gas mixtures was shown to influence spoilage occurrence. The collected data clearly reveal a potentially protective role of lactic acid bacteria. Besides, while a spoilage role could be attributed to Pseudomonas spp., the growth of mesophilic aerobic microbes, Brochothrix spp. and Enterobacteriaceae seemed independent of spoilage occurrence time.