The gut virome is associated with stress-induced changes in behaviour and immune responses in mice.

The microbiota-gut-brain axis has been shown to play an important role in the stress response, but previous work has focused primarily on the role of the bacteriome. The gut virome constitutes a major portion of the microbiome, with bacteriophages having the potential to remodel bacteriome structure and activity. Here we use a mouse model of chronic social stress, and employ 16S rRNA and whole metagenomic sequencing on faecal pellets to determine how the virome is modulated by and contributes to the effects of stress. We found that chronic stress led to behavioural, immune and bacteriome alterations in mice that were associated with changes in the bacteriophage class Caudoviricetes and unassigned viral taxa. To determine whether these changes were causally related to stress-associated behavioural or physiological outcomes, we conducted a faecal virome transplant from mice before stress and autochthonously transferred it to mice undergoing chronic social stress. The transfer of the faecal virome protected against stress-associated behaviour sequelae and restored stress-induced changes in select circulating immune cell populations, cytokine release, bacteriome alterations and gene expression in the amygdala. These data provide evidence that the virome plays a role in the modulation of the microbiota-gut-brain axis during stress, indicating that these viral populations should be considered when designing future microbiome-directed therapies.

Phages Shape Microbial Dynamics and Metabolism of a Model Community Mimicking Cider, a Fermented Beverage.

Model microbial communities are often studied to better understand interactions and fluxes during fermentation processes. However, models that take into account the potential impact of bacteriophages (phages), which are recognized as drivers of microbial communities, are scarce, especially in fermented foods. This study aimed at investigating the behavior of a cider model microbial community, which was subjected to disturbance in the presence or absence of phages and at two different temperatures (25 °C and 15 °C). The model microbial community was composed of three lactic acid bacteria (LAB) strains belonging to the species Liquorilactobacillus mali, Leuconostoc mesenteroides and Oenococcus oeni, and of a Saccharomyces uvarum yeast strain. Two phages were selected, targeting L. mali and Ln. mesenteroides strains. In order to follow the behavior of the microbial community model, the phages and microbial strains were enumerated at several time points, and the metabolic signatures (sugar consumption, production of organic acids and volatile organic compounds) of the model microbial community were monitored. At 25 °C, the community with phages (P) was significantly closer to the control condition (C) than to the condition without phages (D). Microbial levels were similar between conditions C and P, which were characterized by high concentrations of compounds such as 2-phenylethanol, ethyl octanoate and isoamyl alcohol, and more globally by a more complex metabolic signature than that of condition D. In condition D, L. mali and Ln. mesenteroides were dominant while S. uvarum and O. oeni were less present, and this condition was characterized by a high concentration of ethyl lactate. At 15 °C, condition P differed from conditions C and D, as Ln. mesenteroides was not detected while the other strains all reached approximately the same levels. The metabolic range of condition P was less important than for conditions C and D. The current study showed that the influence of phages on the model microbial community dynamics and metabolisms after a disturbance phenomenon was temperature-dependent.

Molecular approaches to uncover phage-lactic acid bacteria interactions in a model community simulating fermented beverages.

Food microbial diversity and fluxes during the fermentation processes are well studied whereas phages-bacteria interactions are still poorly described in the literature. This is especially true in fermented beverages, and especially in cider, which is an alcoholic fermented apple beverage. The transcriptomic and proteomic responses of the lactic acid bacterium (LAB) Liquorilactobacillus mali UCMA 16447 to a lytic infection by phage UCMA 21115, both isolated from cider, were investigated, in order to get a better understanding of phages-bacteria interactions in such fermented beverage. During phage infection, 122 and 215 genes were differentially expressed in L. mali UCMA 16447 strain at T15 and T60 respectively, when compared to the uninfected condition. The same trends were confirmed by the proteomic study, with a total of 28 differentially expressed proteins found at T60. Overall, genes encoding cellular functions, such as carbohydrate metabolism, translation, and signal transduction, were downregulated, while genes involved in nucleotide metabolism and in the control of DNA integrity were upregulated in response to phage infection. This work also highlighted that phage infection repressed many genes involved in bacterial cell motility, and affected glycolysis.

Investigation of the Phageome and Prophages in French Cider, a Fermented Beverage.

Phageomes are known to play a key role in the functioning of their associated microbial communities. The phageomes of fermented foods have not been studied thoroughly in fermented foods yet, and even less in fermented beverages. Two approaches were employed to investigate the presence of phages in cider, a fermented beverage made from apple, during a fermentation process of two cider tanks, one from an industrial producer and one from a hand-crafted producer. The phageome (free lytic phages) was explored in cider samples with several methodological developments for total phage DNA extraction, along with single phage isolation. Concentration methods, such as tangential flow filtration, flocculation and classical phage concentration methods, were employed and tested to extract free phage particles from cider. This part of the work revealed a very low occurrence of free lytic phage particles in cider. In parallel, a prophage investigation during the fermentation process was also performed using a metagenomic approach on the total bacterial genomic DNA. Prophages in bacterial metagenomes in the two cider tanks seemed also to occur in low abundance, as a total of 1174 putative prophages were identified in the two tanks overtime, and only two complete prophages were revealed. Prophage occurrence was greater at the industrial producer than at the hand-crafted producer, and different dynamics of prophage trends were also observed during fermentation. This is the first report dealing with the investigation of the phageome and of prophages throughout a fermentation process of a fermented beverage.

Phage community involvement in fermented beverages: an open door to technological advances?

Bacteriophages (phages) are considered the most abundant biological entities on Earth. An increasing interest in understanding phage communities, also called viromes or phageomes, has arisen over the past decade especially thanks to the development and the accessibility of Next Generation Sequencing techniques. Despite the increasing amount of available metagenomic data on microbial communities in various habitats, viromes remain poorly described in the scientific literature particularly when it comes to fermented food and beverages such as wine and cider. In this review, a particular attention is paid to the current knowledge on phage communities, with a special focus on fermented food viromes and the methodological tools available to undertake their study. There is a striking lack of available data on the fermented foods and beverages viromes. As far as we know, and although a number of phages have been isolated from wine, no general study has to date been carried out to assess the diversity of viromes in fermented beverages and their possible interactions with microbiota throughout the fermentation process. With the aim of establishing connections between the currently used technologies to carry out the analysis of viromes, possible applications of current knowledge to fermented beverages are examined.

Autochthonous faecal viral transfer (FVT) impacts the murine microbiome after antibiotic perturbation.

BACKGROUND: It has become increasingly accepted that establishing and maintaining a complex and diverse gut microbiota is fundamental to human health. There are growing efforts to identify means of modulating and influencing the microbiota, especially in individuals who have experienced a disruption in their native microbiota. Faecal microbiota transplantation (FMT) is one method that restores diversity to the microbiota of an individual by introducing microbes from a healthy donor. FMT introduces the total microbial load into the recipient, including the bacteria, archaea, yeasts, protists and viruses. In this study, we investigated whether an autochthonous faecal viral transfer (FVT), in the form of a sterile faecal filtrate, could impact the recovery of a bacteriome disrupted by antibiotic treatment. RESULTS: Following antibiotic disruption of the bacteriome, test mice received an FVT harvested prior to antibiotic treatment, while control mice received a heat- and nuclease-treated FVT. In both groups of mice, the perturbed microbiome reverted over time to one more similar to the pre-treatment one. However, the bacteriomes of mice that received an FVT, in which bacteriophages predominate, separated from those of the control mice as determined by principal co-ordinate analysis (PCoA). Moreover, analysis of the differentially abundant taxa indicated a closer resemblance to the pre-treatment bacteriome in the test mice that had received an FVT. Similarly, metagenomic sequencing of the virome confirmed that faecal bacteriophages of FVT and control mice differed over time in both abundance and diversity, with the phages constituting the FVT persisting in mice that received them. CONCLUSIONS: An autochthonous virome transfer reshaped the bacteriomes of mice post-antibiotic treatment such that they more closely resembled the pre-antibiotic microbiota profile compared to mice that received non-viable phages. Thus, FVT may have a role in addressing antibiotic-associated microbiota alterations and potentially prevent the establishment of post-antibiotic infection. Given that bacteriophages are biologically inert in the absence of their host bacteria, they could form a safe and effective alternative to whole microbiota transplants that could be delivered during/following perturbation of the gut flora.

Oenococcus sicerae sp. nov., isolated from French cider.

Two Gram-stain-positive, small ellipsoidal cocci, non-motile, oxidase- and catalase-negative, and facultative anaerobic strains (UCMA15228T and UCMA17102) were isolated in France, from fermented apple juices (ciders). The 16S rRNA gene sequence was identical between the two isolates and showed 97 % similarity with respect to the closest related species Oenococcus oeni and O. kitaharae. Therefore, the two isolates were classified within the genus Oenococcus. The phylogeny based on the pheS gene sequences also confirmed the position of the new taxon. DNA-DNA hybridizations based on in silico genome-to-genome comparisons (GGDC) and Average Nucleotide Identity (ANI) values, as well as species-specific PCR, validated the novelty of the taxon. Various phenotypic characteristics such as the optimum temperature and pH for growth, the ability to metabolise sugars, the aptitude to perform the malolactic fermentation, and the resistance to ethanol and NaCl, revealed that the two strains are distinguishable from the other members of the Oenococcus genus. The combined genotypic and phenotypic data support the classification of strains UCMA15228T and UCMA17102 into a novel species of Oenococcus, for which the name O. sicerae sp. nov. is proposed. The type strain is UCMA15228T (=DSM107163T=CIRM-BIA2288T).

Phages for biocontrol in foods: What opportunities for Salmonella sp. control along the dairy food chain?

Controlling the presence of pathogenic bacteria, such as Salmonella sp., in dairy products production is a burning issue since contamination with Salmonella can occur at any stage of the production chain. The use of Salmonella-phages applied as control agents has gained considerable interest. Nonetheless, Salmonella-phage applications specifically intended for ensuring the safety of dairy products are scarce. This review identifies recent advances in the use of Salmonella-phages that are or could be applied along the dairy food chain, in a farm-to-fork approach. Salmonella-phages can be promising tools to reduce the shedding of Salmonella in cattle, and to reduce and control Salmonella occurrence in postharvest food (such as food additives), and in food processing facilities (such as biosanitizing agents). These control measures, combined with existing methods and other biocontrol agents, constitute new opportunities to reduce Salmonella occurrence along the dairy food production, and consequently to alleviate the risk of Salmonella contamination in dairy products.

Multiplex PCR for rapid identification of major lactic acid bacteria genera in cider and other fermented foods.

Lactobacillus, Pediococcus, Oenococcus and Leuconostoc are the main Lactic Acid Bacteria (LAB) genera present in cider as they are able to survive this hostile environment. LAB play a significant role in cider quality, for example in the process of malolactic fermentation, even though they can also be involved in spoilage of cider (production of biogenic amines, exopolysaccharides, off-flavours…). In this context a better monitoring of the fermentation process is a matter of interest to guarantee cider quality. In the present study, we designed a genus-specific multiplex PCR for a rapid and simultaneous detection of the four main LAB genera involved in cider production. This multiplex PCR worked equally with purified genomic DNA of bacterial isolates and with colonies directly picked from agar plates. This new PCR method was also successfully extended to wine and dairy isolates, and thus constitutes an effective tool to quickly identify LAB associated with fermented foods. Moreover, many biodiversity studies would also benefit from this fast, cheap and reliable identification method.

Virulence Gene Sequencing Highlights Similarities and Differences in Sequences in Listeria monocytogenes Serotype 1/2a and 4b Strains of Clinical and Food Origin From 3 Different Geographic Locations.

The prfA-virulence gene cluster (pVGC) is the main pathogenicity island in Listeria monocytogenes, comprising the prfA, plcA, hly, mpl, actA, and plcB genes. In this study, the pVGC of 36 L. monocytogenes isolates with respect to different serotypes (1/2a or 4b), geographical origin (Australia, Greece or Ireland) and isolation source (food-associated or clinical) was characterized. The most conserved genes were prfA and hly, with the lowest nucleotide diversity (π) among all genes (P < 0.05), and the lowest number of alleles, substitutions and non-synonymous substitutions for prfA. Conversely, the most diverse gene was actA, which presented the highest number of alleles (n = 20) and showed the highest nucleotide diversity. Grouping by serotype had a significantly lower π value (P < 0.0001) compared to isolation source or geographical origin, suggesting a distinct and well-defined unit compared to other groupings. Among all tested genes, only hly and mpl were those with lower nucleotide diversity in 1/2a serotype than 4b serotype, reflecting a high within-1/2a serotype divergence compared to 4b serotype. Geographical divergence was noted with respect to the hly gene, where serotype 4b Irish strains were distinct from Greek and Australian strains. Australian strains showed less diversity in plcB and mpl relative to Irish or Greek strains. Notable differences regarding sequence mutations were identified between food-associated and clinical isolates in prfA, actA, and plcB sequences. Overall, these results indicate that virulence genes follow different evolutionary pathways, which are affected by a strain's origin and serotype and may influence virulence and/or epidemiological dominance of certain subgroups.

Viromes of one year old infants reveal the impact of birth mode on microbiome diversity.

Establishing a diverse gut microbiota after birth is being increasingly recognised as important for preventing illnesses later in life. It is well established that bacterial diversity rapidly increases post-partum; however, few studies have examined the infant gut virome/phageome during this developmental period. We performed a metagenomic analysis of 20 infant faecal viromes at one year of age to determine whether spontaneous vaginal delivery (SVD) or caesarean section (CS) influenced viral composition. We find that birth mode results in distinctly different viral communities, with SVD infants having greater viral and bacteriophage diversity. We demonstrate that CrAssphage is acquired early in life, both in this cohort and two others, although no difference in birth mode is detected. A previous study has shown that bacterial OTU's (operational taxonomic units) identified in the same infants could not discriminate between birth mode at 12 months of age. Therefore, our results indicate that vertical transmission of viral communities from mother to child may play a role in shaping the early life microbiome, and that birth mode should be considered when studying the early life gut virome.

Reproducible protocols for metagenomic analysis of human faecal phageomes.

BACKGROUND: Recent studies have demonstrated that the human gut is populated by complex, highly individual and stable communities of viruses, the majority of which are bacteriophages. While disease-specific alterations in the gut phageome have been observed in IBD, AIDS and acute malnutrition, the human gut phageome remains poorly characterised. One important obstacle in metagenomic studies of the human gut phageome is a high level of discrepancy between results obtained by different research groups. This is often due to the use of different protocols for enriching virus-like particles, nucleic acid purification and sequencing. The goal of the present study is to develop a relatively simple, reproducible and cost-efficient protocol for the extraction of viral nucleic acids from human faecal samples, suitable for high-throughput studies. We also analyse the effect of certain potential confounding factors, such as storage conditions, repeated freeze-thaw cycles, and operator bias on the resultant phageome profile. Additionally, spiking of faecal samples with an exogenous phage standard was employed to quantitatively analyse phageomes following metagenomic sequencing. Comparative analysis of phageome profiles to bacteriome profiles was also performed following 16S rRNA amplicon sequencing. RESULTS: Faecal phageome profiles exhibit an overall greater individual specificity when compared to bacteriome profiles. The phageome and bacteriome both exhibited moderate change when stored at + 4 °C or room temperature. Phageome profiles were less impacted by multiple freeze-thaw cycles than bacteriome profiles, but there was a greater chance for operator effect in phageome processing. The successful spiking of faecal samples with exogenous bacteriophage demonstrated large variations in the total viral load between individual samples. CONCLUSIONS: The faecal phageome sequencing protocol developed in this study provides a valuable additional view of the human gut microbiota that is complementary to 16S amplicon sequencing and/or metagenomic sequencing of total faecal DNA. The protocol was optimised for several confounding factors that are encountered while processing faecal samples, to reduce discrepancies observed within and between research groups studying the human gut phageome. Rapid storage, limited freeze-thaw cycling and spiking of faecal samples with an exogenous phage standard are recommended for optimum results.

A long and abundant non-coding RNA in Lactobacillus salivarius.

Lactobacillus salivarius, found in the intestinal microbiota of humans and animals, is studied as an example of the sub-dominant intestinal commensals that may impart benefits upon their host. Strains typically harbour at least one megaplasmid that encodes functions contributing to contingency metabolism and environmental adaptation. RNA sequencing (RNA-seq)transcriptomic analysis of L. salivarius strain UCC118 identified the presence of a novel unusually abundant long non-coding RNA (lncRNA) encoded by the megaplasmid, and which represented more than 75 % of the total RNA-seq reads after depletion of rRNA species. The expression level of this 520 nt lncRNA in L. salivarius UCC118 exceeded that of the 16S rRNA, it accumulated during growth, was very stable over time and was also expressed during intestinal transit in a mouse. This lncRNA sequence is specific to the L. salivarius species; however, among 45 L. salivarius genomes analysed, not all (only 34) harboured the sequence for the lncRNA. This lncRNA was produced in 27 tested L. salivarius strains, but at strain-specific expression levels. High-level lncRNA expression correlated with high megaplasmid copy number. Transcriptome analysis of a deletion mutant lacking this lncRNA identified altered expression levels of genes in a number of pathways, but a definitive function of this new lncRNA was not identified. This lncRNA presents distinctive and unique properties, and suggests potential basic and applied scientific developments of this phenomenon.

Microorganisms in Fermented Apple Beverages: Current Knowledge and Future Directions.

Production of fermented apple beverages is spread all around the world with specificities in each country. 'French ciders' refer to fermented apple juice mainly produced in the northwest of France and often associated with short periods of consumption. Research articles on this kind of product are scarce compared to wine, especially on phenomena associated with microbial activities. The wine fermentation microbiome and its dynamics, organoleptic improvement for healthy and pleasant products and development of starters are now widely studied. Even if both beverages seem close in terms of microbiome and process (with both alcoholic and malolactic fermentations), the inherent properties of the raw materials and different production and environmental parameters make research on the specificities of apple fermentation beverages worthwhile. This review summarizes current knowledge on the cider microbial ecosystem, associated activities and the influence of process parameters. In addition, available data on cider quality and safety is reviewed. Finally, we focus on the future role of lactic acid bacteria and yeasts in the development of even better or new beverages made from apples.

Three New Escherichia coli Phages from the Human Gut Show Promising Potential for Phage Therapy.

With the emergence of multi-drug resistant bacteria the use of bacteriophages (phages) is gaining renewed interest as promising anti-microbial agents. The aim of this study was to isolate and characterize phages from human fecal samples. Three new coliphages, ɸAPCEc01, ɸAPCEc02 and ɸAPCEc03, were isolated. Their phenotypic and genomic characteristics, and lytic activity against biofilm, and in combination with ciprofloxacin, were investigated. All three phages reduced the growth of E. coli strain DPC6051 at multiplicity of infection (MOI) between 10-3 and 105. A cocktail of all three phages completely inhibited the growth of E. coli. The phage cocktail also reduced biofilm formation and prevented the emergence of phage-resistant mutants which occurred with single phage. When combined with ciprofloxacin, phage alone or in cocktail inhibited the growth of E. coli and prevented the emergence of resistant mutants. These three new phages are promising biocontrol agents for E. coli infections.

Isolation of a Novel Phage with Activity against Streptococcus mutans Biofilms.

Streptococcus mutans is one of the principal agents of caries formation mainly, because of its ability to form biofilms at the tooth surface. Bacteriophages (phages) are promising antimicrobial agents that could be used to prevent or treat caries formation by S. mutans. The aim of this study was to isolate new S. mutans phages and to characterize their antimicrobial properties. A new phage, ɸAPCM01, was isolated from a human saliva sample. Its genome was closely related to the only two other available S. mutans phage genomes, M102 and M102AD. ɸAPCM01 inhibited the growth of S. mutans strain DPC6143 within hours in broth and in artificial saliva at multiplicity of infections as low as 2.5x10-5. In the presence of phage ɸAPCM01 the metabolic activity of a S. mutans biofilm was reduced after 24 h of contact and did not increased again after 48 h, and the live cells in the biofilm decreased by at least 5 log cfu/ml. Despite its narrow host range, this newly isolated S. mutans phage exhibits promising antimicrobial properties.

PFGE as a tool to track Listeria monocytogenes in food processing facilities: case studies.

The use of PFGE to track Listeria monocytogenes strains from the food processing environment to the food is explained in this chapter through two case studies. This illustrates the usefulness of this method to identify putative routes of contamination and persistent strains and to help in the implementation of corrective actions in food processing facilities to control the occurrence of this pathogenic bacterium.

Pulsed-field gel electrophoresis for Leuconostoc mesenteroides and L. pseudomesenteroides.

Pulsed-field gel electrophoresis (PFGE) is a technique using alternating electric fields to help the separation of high molecular weight DNA fragments with a high resolution. This method consists of the digestion of bacterial chromosomal DNA with rare-cutting restriction enzymes and of applying an alternating electrical current between spatially distinct pairs of electrodes. DNA molecules migrate at different speeds according to the size of the fragment. This method is considered as the "gold standard" for genotyping, genetic fingerprinting, epidemiological studies, genome size estimation, and studying radiation-induced DNA damage and repair.

Pulsed field gel electrophoresis for dairy propionibacteria.

Pulsed field gel electrophoresis (PFGE) is a technique using alternating electric fields to migrate high molecular weight DNA fragments with a high resolution. This method consists of the digestion of bacterial chromosomal DNA with rare-cutting restriction enzymes and in applying an alternating electrical current between spatially distinct pairs of electrodes. DNA molecules migrate at different speeds according to the size of the fragments. Among other things, this technique is considered as the "gold standard" for genotyping, genetic fingerprinting, epidemiological studies, genome size estimation, and studying radiation-induced DNA damage and repair. This chapter describes a PFGE method that can be used to differentiate dairy propionibacteria.

Farm to Fork Quantitative Risk Assessment of Listeria monocytogenes Contamination in Raw and Pasteurized Milk Cheese in Ireland.

The objective of this study was to model and quantify the level of Listeria monocytogenes in raw milk cheese (RMc) and pasteurized milk cheese (PMc) from farm to fork using a Bayesian inference approach combined with a quantitative risk assessment. The modeling approach included a prediction of contamination arising from the farm environment as well from cross-contamination within the cheese-processing facility through storage and subsequent human exposure. The model predicted a high concentration of L. monocytogenes in contaminated RMc (mean 2.19 log10 CFU/g) compared to PMc (mean -1.73 log10 CFU/g). The mean probability of illness (P1 for low-risk population, LR) and (P2 for high-risk population, HR, e.g., immunocompromised) adult Irish consumers following exposure to contaminated cheese was 7 × 10(-8) (P1 ) and 9 × 10(-4) (P2 ) for RMc and 7 × 10(-10) (P1 ) and 8 × 10(-6) (P2 ) for PMc, respectively. In addition, the model was used to evaluate performance objectives at various stages, namely, the cheese making and ripening stages, and to set a food safety objective at the time of consumption. A scenario analysis predicted various probabilities of L. monocytogenes contamination along the cheese-processing chain for both RMc and PMc. The sensitivity analysis showed the critical factors for both cheeses were the serving size of the cheese, storage time, and temperature at the distribution stage. The developed model will allow food processors and policymakers to identify the possible routes of contamination along the cheese-processing chain and to reduce the risk posed to human health.