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Designing bacterial co-cultures adapted to ferment mixes of vegetal and animal resources for food diversification and sustainability is becoming a challenge. Among bacteria used in food fermentation, lactic acid bacteria (LAB) are good candidates, as they are used as starter or adjunct in numerous fermented foods, where they allow preservation, enhanced digestibility, and improved flavor. We developed here a strategy to design LAB co-cultures able to ferment a new food made of bovine milk and lupin flour, consisting in: (i) in silico preselection of LAB species for targeted carbohydrate degradation; (ii) in vitro screening of 97 strains of the selected species for their ability to ferment carbohydrates and hydrolyze proteins from milk and lupin and clustering strains that displayed similar phenotypes; and (iii) assembling strains randomly sampled from clusters that showed complementary phenotypes. The designed co-cultures successfully expressed the targeted traits i.e., hydrolyzed proteins and degraded raffinose family oligosaccharides of lupin and lactose of milk in a large range of concentrations. They also reduced an off-flavor-generating volatile, hexanal, and produced various desirable flavor compounds. Most of the strains in co-cultures achieved higher cell counts than in monoculture, suggesting positive interactions. This work opens new avenues for the development of innovative fermented food products based on functionally complementary strains in the worldwide context of diet diversification.

Designing bacterial co-cultures adapted to ferment mixes of vegetal and animal resources for food diversification and sustainability is becoming a challenge. Among bacteria used in food fermentation, lactic acid bacteria (LAB) are good candidates, as they are used as starter or adjunct in numerous fermented foods, where they allow preservation, enhanced digestibility, and improved flavor. We developed here a strategy to design LAB co-cultures able to ferment a new food made of bovine milk and lupin flour, consisting in: (i) in silico preselection of LAB species for targeted carbohydrate degradation; (ii) in vitro screening of 97 strains of the selected species for their ability to ferment carbohydrates and hydrolyze proteins from milk and lupin and clustering strains that displayed similar phenotypes; and (iii) assembling strains randomly sampled from clusters that showed complementary phenotypes. The designed co-cultures successfully expressed the targeted traits i.e., hydrolyzed proteins and degraded raffinose family oligosaccharides of lupin and lactose of milk in a large range of concentrations. They also reduced an off-flavor-generating volatile, hexanal, and produced various desirable flavor compounds. Most of the strains in co-cultures achieved higher cell counts than in monoculture, suggesting positive interactions. This work opens new avenues for the development of innovative fermented food products based on functionally complementary strains in the worldwide context of diet diversification.

Streptococcus salivarius is a significant contributor to the human oral, pharyngeal and gut microbiomes that contribute to the maintenance of health. The high genomic diversity observed in this species is mainly caused by horizontal gene transfer. This work aimed to evaluate the contribution of integrative and conjugative elements (ICEs) and integrative and mobilizable elements (IMEs) in S. salivarius genome diversity. For this purpose, we performed an in-depth analysis of 75 genomes of S. salivarius and searched for signature genes of conjugative and mobilizable elements. This analysis led to the retrieval of 69 ICEs, 165 IMEs and many decayed elements showing their high prevalence in S. salivarius genomes. The identification of almost all ICE and IME boundaries allowed the identification of the genes in which these elements are inserted. Furthermore, the exhaustive analysis of the adaptation genes carried by these elements showed that they encode numerous functions such as resistance to stress, to antibiotics or to toxic compounds, and numerous enzymes involved in diverse cellular metabolic pathways. These data support the idea that not only ICEs but also IMEs and decayed elements play an important role in S. salivarius adaptation to the environment.

Streptococcus salivarius is a significant contributor to the human oral, pharyngeal and gut microbiomes that contribute to the maintenance of health. The high genomic diversity observed in this species is mainly caused by horizontal gene transfer. This work aimed to evaluate the contribution of integrative and conjugative elements (ICEs) and integrative and mobilizable elements (IMEs) in S. salivarius genome diversity. For this purpose, we performed an in-depth analysis of 75 genomes of S. salivarius and searched for signature genes of conjugative and mobilizable elements. This analysis led to the retrieval of 69 ICEs, 165 IMEs and many decayed elements showing their high prevalence in S. salivarius genomes. The identification of almost all ICE and IME boundaries allowed the identification of the genes in which these elements are inserted. Furthermore, the exhaustive analysis of the adaptation genes carried by these elements showed that they encode numerous functions such as resistance to stress, to antibiotics or to toxic compounds, and numerous enzymes involved in diverse cellular metabolic pathways. These data support the idea that not only ICEs but also IMEs and decayed elements play an important role in S. salivarius adaptation to the environment.

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.

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.

Background The European Community has adopted very restrictive policies regarding the dissemination and use of genetically modified organisms (GMOs). In fact, a maximum threshold of 0.9% of contaminating GMOs is tolerated for a "GMO-free" label. In recent years, imports of undescribed GMOs have been detected. Their sequences are not described and therefore not detectable by conventional approaches, such as PCR. Results We developed DUGMO, a bioinformatics pipeline for the detection of genetically modified (GM) bacteria, including unknown GM bacteria, based on Illumina paired-end sequencing data. The method is currently focused on the detection of GM bacteria with - possibly partial - transgenes in pure bacterial samples. In the preliminary steps, coding sequences (CDSs) are aligned through two successive BLASTN against the host pangenome with relevant tuned parameters to discriminate CDSs belonging to the wild type genome (wgCDS) from potential GM coding sequences (pgmCDSs). Then, Bray-Curtis distances are calculated between the wgCDS and each pgmCDS, based on the difference of genomic vocabulary. Finally, two machine learning methods, namely the Random Forest and Generalized Linear Model, are carried out to target true GM CDS(s), based on six variables including Bray-Curtis distances and GC content. Tests carried out on a GMBacillus subtilisshowed 25 positive CDSs corresponding to the chloramphenicol resistance gene and CDSs of the inserted plasmids. On a wild typeB. subtilis, no false positive sequences were detected. Conclusion DUGMO detects exogenous CDS, truncated, fused or highly mutated wild CDSs in high-throughput sequencing data, and was shown to be efficient at detecting GM sequences, but it might also be employed for the identification of recent horizontal gene transfers.

Mycobacterium bovis strain Mb3601 was isolated from the lymph node of an infected bovine in a bovine tuberculosis highly enzoonotic area of Burgundy, France. It was selected to obtain a complete genome for a new clonal complex, mainly constituted by SB0120-spoligotype strains that we propose to name "European 3". It was recently described as "clonal group I" based on whole-genome SNP analysis of 87 French strains. Here we describe the 4,365,068 bp complete genome obtained by the combination of PacBio and Illumina technologies. This genome of 65.64% G + C content includes 4024 predicted protein-coding genes, 52 tRNA, 3 rRNA and 11 copies of IS6110.

Mycobacterium bovis strain Mb3601 was isolated from the lymph node of an infected bovine in a bovine tuberculosis highly enzoonotic area of Burgundy, France. It was selected to obtain a complete genome for a new clonal complex, mainly constituted by SB0120-spoligotype strains that we propose to name "European 3". It was recently described as "clonal group I" based on whole-genome SNP analysis of 87 French strains. Here we describe the 4,365,068 bp complete genome obtained by the combination of PacBio and Illumina technologies. This genome of 65.64% G + C content includes 4024 predicted protein-coding genes, 52 tRNA, 3 rRNA and 11 copies of IS6110.

The developmentof high throughput sequencing (HTS) technologies has substantially improvedanalysis of bacterial community diversity, composition,and functions. Over the last decade, HTS has been used extensively to identify the diversity and composition of tick microbial communities. However, a growing number of studies are warning about the impact of contamination brought along the different steps of the analytical process, from DNA extraction to amplification. In low biomass samples, e.g. individual tick samples, these contaminants may represent a large part of the obtained sequences,and thus generate considerable errors in downstream analyses and in the interpretation of results. Most studies of tick microbiota either do not mention the inclusion of controls during the DNA extraction or amplification steps, or consider the lack of an electrophoresis signal as an absence of contamination. In this context, we aimed to assess theproportion of contaminantsequences resulting from thesesteps. We analyzed the microbiota of individual Ixodesricinusticksbyincluding several categories of controls throughout the analytical process:crushing, DNA extraction,and DNA amplification Results Controls yielded a significant number of sequences (1,126 to 13,198 mean sequences,depending onthe control category). Some operational taxonomic units (OTUs)detected in these controls belong to genera reported in previous tick microbiota studies. Inthis study, these OTUs accounted for 50.9% of the total number of sequences inour samples, and wereconsidered contaminants. Contamination levels (i.e. the percentage of sequences belonging to OTUs identified as contaminants) variedwith tick stage and gender: 76.3% of nymphs and 75% of males demonstrated contamination over 50%, while most females (65.7%) had rateslower than 20%. Contamination mainly correspondedto OTUs detected in crushing and DNA extraction controls, highlighting the importance of carefully controlling these steps. -Conclusion Here,we showed that contaminant OTUs from extraction and amplification stepscan represent more than half the total sequence yield in sequencing runs,and lead to unreliable results when characterizing tick microbial communities.We thus strongly advise the routine use of blanks and negative controls in tick microbiota studies, and more generally in studies involving low biomass.

The developmentof high throughput sequencing (HTS) technologies has substantially improvedanalysis of bacterial community diversity, composition,and functions. Over the last decade, HTS has been used extensively to identify the diversity and composition of tick microbial communities. However, a growing number of studies are warning about the impact of contamination brought along the different steps of the analytical process, from DNA extraction to amplification. In low biomass samples, e.g. individual tick samples, these contaminants may represent a large part of the obtained sequences,and thus generate considerable errors in downstream analyses and in the interpretation of results. Most studies of tick microbiota either do not mention the inclusion of controls during the DNA extraction or amplification steps, or consider the lack of an electrophoresis signal as an absence of contamination. In this context, we aimed to assess theproportion of contaminantsequences resulting from thesesteps. We analyzed the microbiota of individual Ixodesricinusticksbyincluding several categories of controls throughout the analytical process:crushing, DNA extraction,and DNA amplification Results Controls yielded a significant number of sequences (1,126 to 13,198 mean sequences,depending onthe control category). Some operational taxonomic units (OTUs)detected in these controls belong to genera reported in previous tick microbiota studies. Inthis study, these OTUs accounted for 50.9% of the total number of sequences inour samples, and wereconsidered contaminants. Contamination levels (i.e. the percentage of sequences belonging to OTUs identified as contaminants) variedwith tick stage and gender: 76.3% of nymphs and 75% of males demonstrated contamination over 50%, while most females (65.7%) had rateslower than 20%. Contamination mainly correspondedto OTUs detected in crushing and DNA extraction controls, highlighting the importance of carefully controlling these steps. -Conclusion Here,we showed that contaminant OTUs from extraction and amplification stepscan represent more than half the total sequence yield in sequencing runs,and lead to unreliable results when characterizing tick microbial communities.We thus strongly advise the routine use of blanks and negative controls in tick microbiota studies, and more generally in studies involving low biomass.

We consider the problem of incorporating evolutionary information (e.g., taxonomic or phylogenic trees) in the context of metagenomics differential analysis. Recent results published in the literature propose different ways to leverage the tree structure to increase the detection rate of differentially abundant taxa. Here, we propose instead to use a different hierarchical structure, in the form of a correlation-based tree, as it may capture the structure of the data better than the phylogeny. We first show that the correlation tree and the phylogeny are significantly different before turning to the impact of tree choice on detection rates. Using synthetic data, we show that the tree does have an impact: smoothing p-values according to the phylogeny leads to equal or inferior rates as smoothing according to the correlation tree. However, both trees are outperformed by the classical, non-hierarchical, Benjamini-Hochberg (BH) procedure in terms of detection rates. Other procedures may use the hierarchical structure with profit but do not control the False Discovery Rate (FDR) a priori and remain inferior to a classical Benjamini-Hochberg procedure with the same nominal FDR. On real datasets, no hierarchical procedure had significantly higher detection rate that BH. Intuition advocates that the use of hierarchical structures should increase the detection rate of differentially abundant taxa in microbiome studies. However, our results suggest that current hierarchical procedures are still inferior to standard methods and more effective procedures remain to be invented.

We consider the problem of incorporating evolutionary information (e.g., taxonomic or phylogenic trees) in the context of metagenomics differential analysis. Recent results published in the literature propose different ways to leverage the tree structure to increase the detection rate of differentially abundant taxa. Here, we propose instead to use a different hierarchical structure, in the form of a correlation-based tree, as it may capture the structure of the data better than the phylogeny. We first show that the correlation tree and the phylogeny are significantly different before turning to the impact of tree choice on detection rates. Using synthetic data, we show that the tree does have an impact: smoothing p-values according to the phylogeny leads to equal or inferior rates as smoothing according to the correlation tree. However, both trees are outperformed by the classical, non-hierarchical, Benjamini-Hochberg (BH) procedure in terms of detection rates. Other procedures may use the hierarchical structure with profit but do not control the False Discovery Rate (FDR) a priori and remain inferior to a classical Benjamini-Hochberg procedure with the same nominal FDR. On real datasets, no hierarchical procedure had significantly higher detection rate that BH. Intuition advocates that the use of hierarchical structures should increase the detection rate of differentially abundant taxa in microbiome studies. However, our results suggest that current hierarchical procedures are still inferior to standard methods and more effective procedures remain to be invented.

Spring phenology of temperate trees has advanced worldwide in response to global warming. However, increasing temperatures may not necessarily lead to further phenological advance, especially in the warmer latitudes because of insufficient chilling and/or shorter daylength. Determining the start of the forcing phase, i.e. when buds are able to respond to warmer temperatures in spring, is therefore crucial to predict how phenology will change in the future. In this study, we used 4,056 leaf-out date observations during the period 1969-2017 for clones of European beech (Fagus sylvatica L.) and pedunculate oak (Quercus robur L.) planted in 63 sites covering a large latitudinal gradient (from Portugal ~ 41°N to Norway ~ 63°N) at the International Phenological Gardens in order to (i) evaluate how the sensitivity periods to forcing and chilling have changed with climate warming, and (ii) test whether consistent patterns occur along biogeographical gradients, i.e. from colder to warmer environments. Partial Least Squares regressions suggest that the length of the forcing period has been extended over the recent decades with climate warming in the colder latitudes but has been shortened in the warmer latitudes for both species, with a more pronounced shift for beech. We attribute the lengthening of the forcing period in the colder latitudes to earlier opportunities with temperatures that can promote bud development. In contrast, at warmer or oceanic climates, the beginning of the forcing period has been delayed, possibly due to insufficient chilling. However, in spite of a later beginning of the forcing period, spring phenology has continued to advance at these areas due to a faster satisfaction of heat requirements induced by climate warming. Overall, our results support that ongoing climate warming will have different effects on the spring phenology of forest trees across latitudes due to the interactions between chilling and forcing requirements and photoperiod.

Spring phenology of temperate trees has advanced worldwide in response to global warming. However, increasing temperatures may not necessarily lead to further phenological advance, especially in the warmer latitudes because of insufficient chilling and/or shorter daylength. Determining the start of the forcing phase, i.e. when buds are able to respond to warmer temperatures in spring, is therefore crucial to predict how phenology will change in the future. In this study, we used 4,056 leaf-out date observations during the period 1969-2017 for clones of European beech (Fagus sylvatica L.) and pedunculate oak (Quercus robur L.) planted in 63 sites covering a large latitudinal gradient (from Portugal ~ 41°N to Norway ~ 63°N) at the International Phenological Gardens in order to (i) evaluate how the sensitivity periods to forcing and chilling have changed with climate warming, and (ii) test whether consistent patterns occur along biogeographical gradients, i.e. from colder to warmer environments. Partial Least Squares regressions suggest that the length of the forcing period has been extended over the recent decades with climate warming in the colder latitudes but has been shortened in the warmer latitudes for both species, with a more pronounced shift for beech. We attribute the lengthening of the forcing period in the colder latitudes to earlier opportunities with temperatures that can promote bud development. In contrast, at warmer or oceanic climates, the beginning of the forcing period has been delayed, possibly due to insufficient chilling. However, in spite of a later beginning of the forcing period, spring phenology has continued to advance at these areas due to a faster satisfaction of heat requirements induced by climate warming. Overall, our results support that ongoing climate warming will have different effects on the spring phenology of forest trees across latitudes due to the interactions between chilling and forcing requirements and photoperiod.

Despite an overall temporal stability in time of the human gut microbiota at the phylum level, strong variations in species abundance have been observed. We are far from a clear understanding of what promotes or disrupts the stability of microbiome communities. Environmental factors, like food or antibiotic use, modify the gut microbiota composition, but their overall impacts remain relatively low. Phages, the viruses that infect bacteria, might constitute important factors explaining temporal variations in species abundance. Gut bacteria harbour numerous prophages, or dormant viruses, which can evolve to become ultravirulent phage mutants, potentially leading to important bacterial death. Whether such phenomenon occurs in the mammal’s microbiota has been largely unexplored. Here we studied temperate phage–bacteria coevolution in gnotoxenic mice colonised with Roseburia intestinalis, a dominant symbiont of the human gut microbiota, and Escherichia coli, a sub-dominant member of the same microbiota. We show that R. intestinalis L1-82 harbours two active prophages, Jekyll and Shimadzu. We observed the systematic evolution in mice of ultravirulent Shimadzu phage mutants, which led to a collapse of R. intestinalis population. In a second step, phage infection drove the fast counterevolution of host phage resistance mainly through phage-derived spacer acquisition in a clustered regularly interspaced short palindromic repeats array. Alternatively, phage resistance was conferred by a prophage originating from an ultravirulent phage with a restored ability to lysogenize. Our results demonstrate that prophages are a potential source of ultravirulent phages that can successfully infect most of the susceptible bacteria. This suggests that prophages can play important roles in the short-term temporal variations observed in the composition of the gut microbiota.

Despite an overall temporal stability in time of the human gut microbiota at the phylum level, strong variations in species abundance have been observed. We are far from a clear understanding of what promotes or disrupts the stability of microbiome communities. Environmental factors, like food or antibiotic use, modify the gut microbiota composition, but their overall impacts remain relatively low. Phages, the viruses that infect bacteria, might constitute important factors explaining temporal variations in species abundance. Gut bacteria harbour numerous prophages, or dormant viruses, which can evolve to become ultravirulent phage mutants, potentially leading to important bacterial death. Whether such phenomenon occurs in the mammal’s microbiota has been largely unexplored. Here we studied temperate phage–bacteria coevolution in gnotoxenic mice colonised with Roseburia intestinalis, a dominant symbiont of the human gut microbiota, and Escherichia coli, a sub-dominant member of the same microbiota. We show that R. intestinalis L1-82 harbours two active prophages, Jekyll and Shimadzu. We observed the systematic evolution in mice of ultravirulent Shimadzu phage mutants, which led to a collapse of R. intestinalis population. In a second step, phage infection drove the fast counterevolution of host phage resistance mainly through phage-derived spacer acquisition in a clustered regularly interspaced short palindromic repeats array. Alternatively, phage resistance was conferred by a prophage originating from an ultravirulent phage with a restored ability to lysogenize. Our results demonstrate that prophages are a potential source of ultravirulent phages that can successfully infect most of the susceptible bacteria. This suggests that prophages can play important roles in the short-term temporal variations observed in the composition of the gut microbiota.

Despite an overall temporal stability in time of the human gut microbiota at the phylum level, strong variations in species abundance have been observed. We are far from a clear understanding of what promotes or disrupts the stability of microbiome communities. Environmental factors, like food or antibiotic use, modify the gut microbiota composition, but their overall impacts remain relatively low. Phages, the viruses that infect bacteria, might constitute important factors explaining temporal variations in species abundance. Gut bacteria harbour numerous prophages, or dormant viruses, which can evolve to become ultravirulent phage mutants, potentially leading to important bacterial death. Whether such phenomenon occurs in the mammal’s microbiota has been largely unexplored. Here we studied temperate phage–bacteria coevolution in gnotoxenic mice colonised with Roseburia intestinalis, a dominant symbiont of the human gut microbiota, and Escherichia coli, a sub-dominant member of the same microbiota. We show that R. intestinalis L1-82 harbours two active prophages, Jekyll and Shimadzu. We observed the systematic evolution in mice of ultravirulent Shimadzu phage mutants, which led to a collapse of R. intestinalis population. In a second step, phage infection drove the fast counterevolution of host phage resistance mainly through phage-derived spacer acquisition in a clustered regularly interspaced short palindromic repeats array. Alternatively, phage resistance was conferred by a prophage originating from an ultravirulent phage with a restored ability to lysogenize. Our results demonstrate that prophages are a potential source of ultravirulent phages that can successfully infect most of the susceptible bacteria. This suggests that prophages can play important roles in the short-term temporal variations observed in the composition of the gut microbiota.

Interactions of diet, gut microbiota, and host genetics play essential roles in the development of metabolic diseases. A/J and C57BL/6J (C57) are two mouse strains known to display different susceptibilities to metabolic disorders. In this context, we analyzed gut microbiota composition in A/J and C57 mice, and assessed its responses to high-fat diet (HFD) and antibiotic (AB) treatment. We also exchanged the gut microbiota between the two strains following AB treatment to evaluate its impact on the metabolism. We showed that A/J and C57 mice have different microbiome structure and composition at baseline. Moreover, A/J and C57 microbiomes responded differently to HFD and AB treatments. Exchange of the gut microbiota between the two strains was successful as recipients’ microbiota resembled donor-strain microbiota. Seven weeks after inoculation, the differences between recipients persisted and were still closer from the donor-strain microbiota. Despite effective microbiota transplants, the response to HFD was not markedly modified in C57 and A/J mice. Particularly, body weight gain and glucose intolerance in response to HFD remained different in the two mouse strains whatever the changes in microbiome composition. This indicated that genetic background has a much stronger impact on metabolic responses to HFD than gut microbiome composition. View