The extent of nitrogen isotopic fractionation in rumen bacteria is associated with changes in rumen nitrogen metabolism.

Nitrogen use efficiency is an important index in ruminants and can be indirectly evaluated through the N isotopic discrimination between the animal and its diet (Δ15Nanimal-diet). The concentration and source of N may determine both the extent of the N isotopic discrimination in bacteria and N use efficiency. We hypothesised that the uptake and release of ammonia by rumen bacteria will affect the natural 15N enrichment of the bacterial biomass over their substrates (Δ15Nbacteria-substrate) and thereby further impacting Δ15Nanimal-diet. To test this hypothesis, two independent in vitro experiments were conducted using two contrasting N sources (organic vs inorganic) at different levels either in pure rumen bacteria culture incubations (Experiment #1) or in mixed rumen cultures (Experiment #2). In Experiment #1, tryptone casein or ammonium chloride were tested at low (1 mM N) and high (11.5 mM N) concentrations on three rumen bacterial strains (Fibrobacter succinogenes, Eubacterium limosum and Xylanibacter ruminicola) incubated in triplicate in anaerobic batch monocultures during 48h. In Experiment #2 mixed rumen cultures were incubated during 120 h with peptone or ammonium chloride at five different levels of N (1.5, 3, 4.5, 6 and 12-mM). In experiment #1, Δ15Nbacteria-substrate was lowest when the ammonia-consumer bacterium Fibrobacter succinogenes was grown on ammonium chloride, and highest when the proteolytic bacterial strain Xylanibacter ruminicola was grown on tryptone. In experiment #2, Δ15Nbacteria-substrate was lower with inorganic (ammonium chloride) vs organic (peptone) N source. A strong negative correlation between Δ15Nbacteria-substrate and Rikenellaceae_RC9_gut_group, a potential fibrolytic rumen bacterium, was detected. Together, our results showed that Δ15Nbacteria-substrate may change according to the balance between synthesis of microbial protein from ammonia versus non-ammonia N sources and confirm the key role of rumen bacteria as modulators of Δ15Nanimal-diet.

Effects of rearing mode on gastro-intestinal microbiota and development, immunocompetence, sanitary status and growth performance of lambs from birth to two months of age.

BACKGROUND: Artificial rearing system, commonly used in prolific sheep breeds, is associated to increased mortality and morbidity rates before weaning, which might be linked to perturbations in digestive tract maturation, including microbiota colonization. This study evaluated the effect of rearing mode (mothered or artificially reared) on the establishment of the rumen and intestinal microbiome of lambs from birth to weaning. We also measured immunological and zootechnical parameters to assess lambs' growth and health. GIT anatomy as well as rumen and intestinal epithelium gene expression were also analysed on weaned animals to assess possible long-term effects of the rearing practice. RESULTS: Total VFA concentrations were higher in mothered lambs at 2 months of age, while artificially-reared lambs had lower average daily gain, a more degraded sanitary status and lower serum IgG concentration in the early growth phase. Metataxonomic analysis revealed higher richness of bacterial and eukaryote populations in mothered vs. artificially-reared lambs in both Rumen and Feces. Beta diversity analysis indicated an evolution of rumen and fecal bacterial communities in mothered lambs with age, not observed in artificially-reared lambs. Important functional microorganisms such as the cellulolytic bacterium Fibrobacter succinogenes and rumen protozoa did not establish correctly before weaning in artificially-reared lambs. Enterobacteriaceae and Escherichia coli were dominant in the fecal microbiota of mothered lambs, but main E. coli virulence genes were not found differential between the two groups, suggesting they are commensal bacteria which could exert a protective effect against pathogens. The fecal microbiota of artificially-reared lambs had a high proportion of lactic acid bacteria taxa. No difference was observed in mucosa gene expression in the two lamb groups after weaning. CONCLUSIONS: The rearing mode influences gastrointestinal microbiota and health-associated parameters in offspring in early life: rumen maturation was impaired in artificially-reared lambs which also presented altered sanitary status and higher risk of gut dysbiosis. The first month of age is thus a critical period where the gastrointestinal tract environment and microbiota are particularly unstable and special care should be taken in the management of artificially fed newborn ruminants.

Dynamic genome-based metabolic modeling of the predominant cellulolytic rumen bacterium Fibrobacter succinogenes S85.

Fibrobacter succinogenes is a cellulolytic bacterium that plays an essential role in the degradation of plant fibers in the rumen ecosystem. It converts cellulose polymers into intracellular glycogen and the fermentation metabolites succinate, acetate, and formate. We developed dynamic models of F. succinogenes S85 metabolism on glucose, cellobiose, and cellulose on the basis of a network reconstruction done with the automatic reconstruction of metabolic model workspace. The reconstruction was based on genome annotation, five template-based orthology methods, gap filling, and manual curation. The metabolic network of F. succinogenes S85 comprises 1,565 reactions with 77% linked to 1,317 genes, 1,586 unique metabolites, and 931 pathways. The network was reduced using the NetRed algorithm and analyzed for the computation of elementary flux modes. A yield analysis was further performed to select a minimal set of macroscopic reactions for each substrate. The accuracy of the models was acceptable in simulating F. succinogenes carbohydrate metabolism with an average coefficient of variation of the root mean squared error of 19%. The resulting models are useful resources for investigating the metabolic capabilities of F. succinogenes S85, including the dynamics of metabolite production. Such an approach is a key step toward the integration of omics microbial information into predictive models of rumen metabolism. IMPORTANCE F. succinogenes S85 is a cellulose-degrading and succinate-producing bacterium. Such functions are central for the rumen ecosystem and are of special interest for several industrial applications. This work illustrates how information of the genome of F. succinogenes can be translated to develop predictive dynamic models of rumen fermentation processes. We expect this approach can be applied to other rumen microbes for producing a model of rumen microbiome that can be used for studying microbial manipulation strategies aimed at enhancing feed utilization and mitigating enteric emissions.

Persistent Circulation of Enterohemorrhagic Escherichia coli (EHEC) O157:H7 in Cattle Farms: Characterization of Enterohemorrhagic Escherichia coli O157:H7 Strains and Fecal Microbial Communities of Bovine Shedders and Non-shedders.

Cattle are carriers, without clinical manifestations, of enterohemorrhagic Escherichia coli (EHEC) O157:H7 responsible for life-threatening infections in humans. A better identification of factors playing a role in maintaining persistence of such strains in cattle is required to develop more effective control measures. Hence, we conducted a study to identify farms with a persistent circulation of EHEC O157:H7. The EHEC O157:H7 herd status of 13 farms, which had previously provided bovine EHEC O157:H7 carriers at slaughter was investigated. Two farms were still housing positive young bulls, and this was true over a 1-year period. Only one fecal sample could be considered from a supershedder, and 60% of the carriers shed concentrations below 10 MPN/g. Moreover, EHEC O157:H7 represented minor subpopulations of E. coli. PFGE analysis of the EHEC O157:H7 strains showed that persistent circulation was due either to the persistence of a few predominant strains or to the repeated exposure of cattle to various strains. Finally, we compared fecal microbial communities of shedders (S) (n = 24) and non-shedders (NS) (n = 28), including 43 young bulls and nine cows, from one farm. Regarding alpha diversity, no significant difference between S vs. NS young bulls (n = 43) was observed. At the genus level, we identified 10 amplicon sequence variant (ASV) indicators of the S or NS groups. The bacterial indicators of S belonged to the family XIII UCG-001, Slackia, and Campylobacter genera, and Ruminococcaceae NK4A21A, Lachnospiraceae-UGC-010, and Lachnospiraceae-GCA-900066575 groups. The NS group indicator ASVs were affiliated to Pirellulaceae-1088-a5 gut group, Anaerovibrio, Victivallis, and Sellimonas genera. In conclusion, the characteristics enhancing the persistence of some predominant strains observed here should be explored further, and studies focused on mechanisms of competition among E. coli strains are also needed.

A live yeast supplementation to gestating ewes improves bioactive molecule composition in colostrum with no impact on its bacterial composition and beneficially affects immune status of the offspring.

Colostrum quality is of paramount importance in the management of optimal ruminant growth and infectious disease prevention in early life. Live yeast supplementation effect during the last month of gestation was evaluated on ewes' colostrum composition. Two groups of ewes (n = 14) carrying twin lambs were constituted and twins were separated into groups (mothered or artificially fed) 12 h after birth. Nutrient, oligosaccharides (OS), IgG and lactoferrin concentrations were measured over 72 h after lambing, and bacterial community was described in colostrum collected at parturition (T0). Immune passive transfer was evaluated through IgG measurement in lamb serum. In both groups, colostral nutrient, OS concentrations and IgG concentrations in colostrum and lamb serum decreased over time (P < 0⋅01), except for lactose, which slightly increased (P < 0⋅001), and lactoferrin, which remained stable. Bacterial population was stable over time with high relative abundances of Aerococcaceae, Corynebacteriaceae, Moraxellaceae and Staphylococcaceae in T0 colostrum. No effect of supplementation was observed in nutrient and lactoferrin concentrations. In supplemented ewes, the level of colostral IgG was higher at T0 and a higher level of serum IgG was observed in lambs born from supplemented mothers and artificially fed, while no effect of supplementation was observed in the mothered lamb groups. Using a metabolomic approach, we showed that supplementation affected OS composition with significantly higher levels of colostral Neu-5Gc compounds up to 5 h after birth. No effect of supplementation was observed on bacterial composition. Our data suggest that live yeast supplementation offsets the negative impact of early separation and incomplete colostrum feeding in neonate lambs.

FI: The Fecobiome Initiative.

Animal husbandry has been key to the sustainability of human societies for millennia. Livestock animals, such as cattle, convert plants to protein biomass due to a compartmentalized gastrointestinal tract (GIT) and the complementary contributions of a diverse GIT microbiota, thereby providing humans with meat and dairy products. Research on cattle gut microbial symbionts has mainly focused on the rumen (which is the primary fermentation compartment) and there is a paucity of functional insight on the intestinal (distal end) microbiota, where most foodborne zoonotic bacteria reside. Here, we present the Fecobiome Initiative (or FI), an international effort that aims at facilitating collaboration on research projects related to the intestinal microbiota, disseminating research results, and increasing public availability of resources. By doing so, the FI can help mitigate foodborne and animal pathogens that threaten livestock and human health, reduce the emergence and spread of antimicrobial resistance in cattle and their proximate environment, and potentially improve the welfare and nutrition of animals. We invite all researchers interested in this type of research to join the FI through our website: www.fecobiome.com.

Transcriptional analysis reveals specific niche factors and response to environmental stresses of enterohemorrhagic Escherichia coli O157:H7 in bovine digestive contents.

BACKGROUND: Enterohemorrhagic Escherichia coli (EHEC) are responsible for severe diseases in humans, and the ruminant digestive tract is considered as their main reservoir. Their excretion in bovine feces leads to the contamination of foods and the environment. Thus, providing knowledge of processes used by EHEC to survive and/or develop all along the bovine gut represents a major step for strategies implementation. RESULTS: We compared the transcriptome of the reference EHEC strain EDL933 incubated in vitro in triplicate samples in sterile bovine rumen, small intestine and rectum contents with that of the strain grown in an artificial medium using RNA-sequencing (RNA-seq), focusing on genes involved in stress response, adhesion systems including the LEE, iron uptake, motility and chemotaxis. We also compared expression of these genes in one digestive content relative to the others. In addition, we quantified short chain fatty acids and metal ions present in the three digestive contents. RNA-seq data first highlighted response of EHEC EDL933 to unfavorable physiochemical conditions encountered during its transit through the bovine gut lumen. Seventy-eight genes involved in stress responses including drug export, oxidative stress and acid resistance/pH adaptation were over-expressed in all the digestive contents compared with artificial medium. However, differences in stress fitness gene expression were observed depending on the digestive segment, suggesting that these differences were due to distinct physiochemical conditions in the bovine digestive contents. EHEC activated genes encoding three toxin/antitoxin systems in rumen content and many gene clusters involved in motility and chemotaxis in rectum contents. Genes involved in iron uptake and utilization were mostly down-regulated in all digestive contents compared with artificial medium, but feo genes were over-expressed in rumen and small intestine compared with rectum. The five LEE operons were more expressed in rectum than in rumen content, and LEE1 was also more expressed in rectum than in small intestine content. CONCLUSION: Our results highlight various strategies that EHEC may implement to survive in the gastrointestinal environment of cattle. These data could also help defining new targets to limit EHEC O157:H7 carriage and shedding by cattle.

Saccharomyces Cerevisiae Var Boulardii CNCM I-1079 Reduces Expression of Genes Involved in Inflammatory Response in Porcine Cells Challenged by Enterotoxigenic E. Coli and Influences Bacterial Communities in an In Vitro Model of the Weaning Piglet Colon.

Enterotoxigenic Escherichia coli (ETEC) is the main infectious agent responsible for piglet post-weaning diarrhea with high mortality rates. Antimicrobials represent the current principal strategy for treating ETEC infections in pig farms, but the occurrence of multi-resistant bacterial strains has considerably increased in the last decades. Thus, finding non-antibiotic alternatives becomes a real emergency. In this context, we investigated the effect of a live yeast strain, Saccharomyces cerevisiae var boulardii CNCM I-1079 (SB) in an in vitro model of the weaning piglet colon implemented with a mucus phase (MPigut-IVM) inoculated with ETEC and coupled with an intestinal porcine cell line IPI-2I. We showed that SB was able to modulate the in vitro microbiota through an increase in Bacteroidiaceae and a decrease in Prevotellaceae families. Effluents collected from the SB treated bioreactors were able to mitigate the expression level of genes encoding non-gel forming mucins, tight junction proteins, innate immune pathway, and pro-inflammatory response in IPI-2I cells. Furthermore, SB exerted a significant protective effect against ETEC adhesion on porcine IPEC-J2 intestinal cells in a dose-dependent manner and showed a positive effect on ETEC-challenged IPEC-J2 by lowering expression of genes involved in pro-inflammatory immune responses. Our results showed that the strain SB CNCM I-1079 could prevent microbiota dysbiosis associated with weaning and protect porcine enterocytes from ETEC infections by reducing bacterial adhesion and modulating the inflammatory response.

Pathogen Challenge and Dietary Shift Alter Microbiota Composition and Activity in a Mucin-Associated in vitro Model of the Piglet Colon (MPigut-IVM) Simulating Weaning Transition.

Enterotoxigenic Escherichia coli (ETEC) is the principal pathogen responsible for post-weaning diarrhea in newly weaned piglets. Expansion of ETEC at weaning is thought to be the consequence of various stress factors such as transient anorexia, dietary change or increase in intestinal inflammation and permeability, but the exact mechanisms remain to be elucidated. As the use of animal experiments raise more and more ethical concerns, we used a recently developed in vitro model of piglet colonic microbiome and mucobiome, the MPigut-IVM, to evaluate the effects of a simulated weaning transition and pathogen challenge at weaning. Our data suggested that the tested factors impacted the composition and functionality of the MPigut-IVM microbiota. The simulation of weaning transition led to an increase in relative abundance of the Prevotellaceae family which was further promoted by the presence of the ETEC strain. In contrast, several beneficial families such as Bacteroidiaceae or Ruminococcaceae and gut health related short chain fatty acids like butyrate or acetate were reduced upon simulated weaning. Moreover, the incubation of MPigut-IVM filtrated effluents with porcine intestinal cell cultures showed that ETEC challenge in the in vitro model led to an increased expression of pro-inflammatory genes by the porcine cells. This study provides insights about the etiology of a dysbiotic microbiota in post-weaning piglets.

Changes in Digestive Microbiota, Rumen Fermentations and Oxidative Stress around Parturition Are Alleviated by Live Yeast Feed Supplementation to Gestating Ewes.

BACKGROUND: In ruminants, physiological and nutritional changes occur peripartum. We investigated if gastro-intestinal microbiota, rumen metabolism and antioxidant status were affected around parturition and what could be the impact of a daily supplementation of a live yeast additive in late gestating ewes. METHODS: Rumen, feces and blood samples were collected from 2 groups of 14 ewes one month and a few days before parturition, and 2 weeks postpartum. RESULTS: In the control ewes close to parturition, slight changes in the ruminal microbiota were observed, with a decrease in the concentration F. succinogenes and in the relative abundance of the Fibrobacteres phylum. Moreover, a decrease in the alpha-diversity of the bacterial community and a reduced relative abundance of the Fibrobacteres phylum were observed in their feces. Control ewes were prone to oxidative stress, as shown by an increase in malondialdehyde (MDA) concentration, a lower total antioxidant status, and higher glutathione peroxidase (GPx) activity in the blood. In the yeast supplemented ewes, most of the microbial changes observed in the control group were alleviated. An increase in GPx activity, and a significant decrease in MDA concentration were measured. CONCLUSIONS: The live yeast used in this study could stabilize gastro-intestinal microbiota and reduce oxidative stress close to parturition.

Weaning-associated feed deprivation stress causes microbiota disruptions in a novel mucin-containing in vitro model of the piglet colon (MPigut-IVM).

BACKGROUND: Risk factors for the etiology of post-weaning diarrhea, a major problem in swine industry associated with enormous economic losses, remain to be fully elucidated. In concordance with the ethical concerns raised by animal experiments, we developed a new in vitro model of the weaning piglet colon (MPigut-IVM) including a mucin bead compartment to reproduce the mucus surface from the gut to which gut microbes can adhere. RESULTS: Our results indicated that the MPigut-IVM is able to establish a representative piglet archaeal and bacterial colon microbiota in terms of taxonomic composition and function. The MPigut-IVM was consequently used to investigate the potential effects of feed deprivation, a common consequence of weaning in piglets, on the microbiota. The lack of nutrients in the MPigut-IVM led to an increased abundance of Prevotellaceae and Escherichia-Shigella and a decrease in Bacteroidiaceae and confirms previous in vivo findings. On top of a strong increase in redox potential, the feed deprivation stress induced modifications of microbial metabolite production such as a decrease in acetate and an increase in proportional valerate, isovalerate and isobutyrate production. CONCLUSIONS: The MPigut-IVM is able to simulate luminal and mucosal piglet microbiota and represent an innovative tool for comparative studies to investigate the impact of weaning stressors on piglet microbiota. Besides, weaning-associated feed deprivation in piglets provokes disruptions of MPigut-IVM microbiota composition and functionality and could be implicated in the onset of post-weaning dysbiosis in piglets.

In Vivo Competitions between Fibrobacter succinogenes, Ruminococcus flavefaciens, and Ruminoccus albus in a Gnotobiotic Sheep Model Revealed by Multi-Omic Analyses.

Fibrobacter succinogenes, Ruminococcus albus, and Ruminococcus flavefaciens are the three predominant cellulolytic bacterial species found in the rumen. In vitro studies have shown that these species compete for adherence to, and growth upon, cellulosic biomass. Yet their molecular interactions in vivo have not heretofore been examined. Gnotobiotically raised lambs harboring a 17-h-old immature microbiota devoid of culturable cellulolytic bacteria and methanogens were inoculated first with F. succinogenes S85 and Methanobrevibacter sp. strain 87.7, and 5 months later, the lambs were inoculated with R. albus 8 and R. flavefaciens FD-1. Longitudinal samples were collected and profiled for population dynamics, gene expression, fibrolytic enzyme activity, in sacco fibrolysis, and metabolite profiling. Quantitative PCR, metagenome and metatranscriptome data show that F. succinogenes establishes at high levels initially but is gradually outcompeted following the introduction of the ruminococci. This shift resulted in an increase in carboxymethyl cellulase (CMCase) and xylanase activities but not in greater fibrolysis, suggesting that F. succinogenes and ruminococci deploy different but equally effective means to degrade plant cell walls. Expression profiles showed that F. succinogenes relied upon outer membrane vesicles and a diverse repertoire of CAZymes, while R. albus and R. flavefaciens preferred type IV pili and either CBM37-harboring or cellulosomal carbohydrate-active enzymes (CAZymes), respectively. The changes in cellulolytics also affected the rumen metabolome, including an increase in acetate and butyrate at the expense of propionate. In conclusion, this study provides the first demonstration of in vivo competition between the three predominant cellulolytic bacteria and provides insight on the influence of these ecological interactions on rumen fibrolytic function and metabolomic response.IMPORTANCE Ruminant animals, including cattle and sheep, depend on their rumen microbiota to digest plant biomass and convert it into absorbable energy. Considering that the extent of meat and milk production depends on the efficiency of the microbiota to deconstruct plant cell walls, the functionality of predominant rumen cellulolytic bacteria, Fibrobacter succinogenes, Ruminococcus albus, and Ruminococcus flavefaciens, has been extensively studied in vitro to obtain a better knowledge of how they operate to hydrolyze polysaccharides and ultimately find ways to enhance animal production. This study provides the first evidence of in vivo competitions between F. succinogenes and the two Ruminococcus species. It shows that a simple disequilibrium within the cellulolytic community has repercussions on the rumen metabolome and fermentation end products. This finding will have to be considered in the future when determining strategies aiming at directing rumen fermentations for animal production.

An Overview of the Elusive Passenger in the Gastrointestinal Tract of Cattle: The Shiga Toxin Producing Escherichia coli.

For approximately 10,000 years, cattle have been our major source of meat and dairy. However, cattle are also a major reservoir for dangerous foodborne pathogens that belong to the Shiga toxin-producing Escherichia coli (STEC) group. Even though STEC infections in humans are rare, they are often lethal, as treatment options are limited. In cattle, STEC infections are typically asymptomatic and STEC is able to survive and persist in the cattle GIT by escaping the immune defenses of the host. Interactions with members of the native gut microbiota can favor or inhibit its persistence in cattle, but research in this direction is still in its infancy. Diet, temperature and season but also industrialized animal husbandry practices have a profound effect on STEC prevalence and the native gut microbiota composition. Thus, exploring the native cattle gut microbiota in depth, its interactions with STEC and the factors that affect them could offer viable solutions against STEC carriage in cattle.

Supplementation of live yeast based feed additive in early life promotes rumen microbial colonization and fibrolytic potential in lambs.

Rumen microbiota is of paramount importance for ruminant digestion efficiency as the microbial fermentations supply the host animal with essential sources of energy and nitrogen. Early separation of newborns from the dam and distribution of artificial milk (Artificial Milking System or AMS) could impair rumen microbial colonization, which would not only affect rumen function but also have possible negative effects on hindgut homeostasis, and impact animal health and performance. In this study, we monitored microbial communities in the rumen and the feces of 16 lambs separated from their dams from 12 h of age and artificially fed with milk replacer and starter feed from d8, in absence or presence of a combination of the live yeast Saccharomyces cerevisiae CNCM I-1077 and selected yeast metabolites. Microbial groups and targeted bacterial species were quantified by qPCR and microbial diversity and composition were assessed by 16S rDNA amplicon sequencing in samples collected from birth to 2 months of age. The fibrolytic potential of the rumen microbiota was analyzed with a DNA microarray targeting genes coding for 8 glycoside hydrolase (GH) families. In Control lambs, poor establishment of fibrolytic communities was observed. Microbial composition shifted as the lambs aged. The live yeast supplement induced significant changes in relative abundances of a few bacterial OTUs across time in the rumen samples, among which some involved in crucial rumen function, and favored establishment of Trichostomatia and Neocallimastigaceae eukaryotic families. The supplemented lambs also harbored greater abundances in Fibrobacter succinogenes after weaning. Microarray data indicated that key cellulase and hemicellulase encoding-genes were present from early age in the rumen and that in the Supplemented lambs, a greater proportion of hemicellulase genes was present. Moreover, a higher proportion of GH genes from ciliate protozoa and fungi was found in the rumen of those animals. This yeast combination improved microbial colonization in the maturing rumen, with a potentially more specialized ecosystem towards efficient fiber degradation, which suggests a possible positive impact on lamb gut development and digestive efficiency.

Microbiota Composition and Functional Profiling Throughout the Gastrointestinal Tract of Commercial Weaning Piglets.

Dietary, environmental, and social stresses induced by weaning transition in pig production are associated with alterations of gut microbiota, diarrhea, and enteric infections. With the boom of -omic technologies, numerous studies have investigated the dynamics of fecal bacterial communities of piglets throughout weaning but much less research has been focused on the composition and functional properties of microbial communities inhabiting other gastrointestinal segments. The objective of the present study was to bring additional information about the piglet bacterial and archaeal microbiota throughout the entire digestive tract, both at the structural level by using quantitative PCR and high-throughput sequencing, and on functionality by measurement of short-chain fatty acids and predictions using Tax4Fun tool. Our results highlighted strong structural and functional differences between microbial communities inhabiting the fore and the lower gut as well as a quantitatively important archaeal community in the hindgut. The presence of opportunistic pathogens was also noticed throughout the entire digestive tract and could trigger infection emergence. Understanding the role of the intestinal piglet microbiota at weaning could provide further information about the etiology of post-weaning infections and lead to the development of effective preventive solutions.

Experimental models to study intestinal microbes-mucus interactions in health and disease.

A close symbiotic relationship exists between the intestinal microbiota and its host. A critical component of gut homeostasis is the presence of a mucus layer covering the gastrointestinal tract. Mucus is a viscoelastic gel at the interface between the luminal content and the host tissue that provides a habitat to the gut microbiota and protects the intestinal epithelium. The review starts by setting up the biological context underpinning the need for experimental models to study gut bacteria-mucus interactions in the digestive environment. We provide an overview of the structure and function of intestinal mucus and mucins, their interactions with intestinal bacteria (including commensal, probiotics and pathogenic microorganisms) and their role in modulating health and disease states. We then describe the characteristics and potentials of experimental models currently available to study the mechanisms underpinning the interaction of mucus with gut microbes, including in vitro, ex vivo and in vivo models. We then discuss the limitations and challenges facing this field of research.

Addressing Global Ruminant Agricultural Challenges Through Understanding the Rumen Microbiome: Past, Present, and Future.

The rumen is a complex ecosystem composed of anaerobic bacteria, protozoa, fungi, methanogenic archaea and phages. These microbes interact closely to breakdown plant material that cannot be digested by humans, whilst providing metabolic energy to the host and, in the case of archaea, producing methane. Consequently, ruminants produce meat and milk, which are rich in high-quality protein, vitamins and minerals, and therefore contribute to food security. As the world population is predicted to reach approximately 9.7 billion by 2050, an increase in ruminant production to satisfy global protein demand is necessary, despite limited land availability, and whilst ensuring environmental impact is minimized. Although challenging, these goals can be met, but depend on our understanding of the rumen microbiome. Attempts to manipulate the rumen microbiome to benefit global agricultural challenges have been ongoing for decades with limited success, mostly due to the lack of a detailed understanding of this microbiome and our limited ability to culture most of these microbes outside the rumen. The potential to manipulate the rumen microbiome and meet global livestock challenges through animal breeding and introduction of dietary interventions during early life have recently emerged as promising new technologies. Our inability to phenotype ruminants in a high-throughput manner has also hampered progress, although the recent increase in "omic" data may allow further development of mathematical models and rumen microbial gene biomarkers as proxies. Advances in computational tools, high-throughput sequencing technologies and cultivation-independent "omics" approaches continue to revolutionize our understanding of the rumen microbiome. This will ultimately provide the knowledge framework needed to solve current and future ruminant livestock challenges.

Transcriptomic analysis reveals specific metabolic pathways of enterohemorrhagic Escherichia coli O157:H7 in bovine digestive contents.

BACKGROUND: The cattle gastrointestinal tract (GIT) is the main enterohemorrhagic Escherichia coli (EHEC) reservoir. In order to identify nutrients required for the survival or multiplication of EHEC in the bovine GIT, we compared the transcriptomes of the EHEC O157:H7 reference strain EDL933 cultured in vitro in bovine digestive contents (DCs) (rumen, small intestine and rectum) using RNA-sequencing. RESULTS: Gene expression profiles showed that EHEC EDL933 activated common but also specific metabolic pathways to survive in the different bovine DCs. Mucus-derived carbohydrates seem important in EHEC nutrition in posterior DCs (small intestine and rectum) but not in rumen content. Additional carbohydrates (xylose, ribose, mannitol, galactitol) as well as gluconeogenic substrates (aspartate, serine, glycerol) would also be used by EHEC as carbon and/or nitrogen sources all along the bovine GIT including the rumen. However, xylose, GalNac, ribose and fucose transport and/or assimilation encoding genes were over-expressed during incubation in rectum content compared with rumen and intestine contents, and genes coding for maltose transport were only induced in rectum. This suggests a role for these carbohydrates in the colonization of the cattle rectum, considered as the major site for EHEC multiplication. In contrast, the transcription of the genes associated with the assimilation of ethanolamine, an important nitrogen source for EHEC, was poorly induced in EHEC growing in rectum content, suggesting that ethanolamine is mainly assimilated in the cattle rumen and small intestine. Respiratory flexibility would also be required for EHEC survival because of the redundancy of dehydrogenases and reductases simultaneously induced in the bovine DCs, probably in response to the availability of electron donors and acceptors. CONCLUSION: EHEC EDL933 showed a high flexibility in the activation of genes involved in respiratory pathways and assimilation of carbon and nitrogen sources, most of them from animal origin. This may allow the bacterium to adapt and survive in the various bovine GIT compartments. Obtaining a better understanding of EHEC physiology in bovine GIT is a key step to ultimately propose strategies to limit EHEC carriage and shedding by cattle.

Aspartate metabolism is involved in the maintenance of enterohaemorrhagic Escherichia coli O157:H7 in bovine intestinal content.

The gastrointestinal tract (GIT) of healthy cattle is the main reservoir of enterohaemorrhagic Escherichia coli (EHEC). Therefore, it is crucial to better understand the physiology of EHEC in the bovine GIT. In this study, we demonstrate that aspartate present in bovine small intestine content (BSIC), was exhausted after incubation of the reference EHEC strain EDL933 but was poorly assimilated by the endogenous microbiota. Furthermore, the bovine commensal E. coli strain BG1 appeared less efficient than EDL933 in aspartate assimilation suggesting a competitive ability of EHEC to assimilate this amino acid. Our results strongly suggest that aspartate, internalized via the DcuA aspartate: succinate antiporting system, is then converted to fumarate and carbamoyl-aspartate, the precursor for UMP biosynthesis. Aspartate assimilation by these two pathways conferred a competitive growth advantage to EHEC in BSIC. In summary, supply of intracellular fumarate due to aspartate deamination and used as an electron acceptor for anaerobic fumarate respiration, as well as de novo synthesis of pyrimidine from aspartate appear to be important pathways favouring EHEC persistence in the bovine gut. Aspartate probably represents an ecological niche for EHEC in the bovine small intestine.

Factors Involved in the Persistence of a Shiga Toxin-Producing Escherichia coli O157:H7 Strain in Bovine Feces and Gastro-Intestinal Content.

Healthy cattle are the primary reservoir for O157:H7 Shiga toxin-producing E. coli responsible for human food-borne infections. Because farm environment acts as a source of cattle contamination, it is important to better understand the factors controlling the persistence of E. coli O157:H7 outside the bovine gut. The E. coli O157:H7 strain MC2, identified as a persistent strain in French farms, possessed the characteristics required to cause human infections and genetic markers associated with clinical O157:H7 isolates. Therefore, the capacity of E. coli MC2 to survive during its transit through the bovine gastro-intestinal tract (GIT) and to respond to stresses potentially encountered in extra-intestinal environments was analyzed. E. coli MC2 survived in rumen fluids, grew in the content of posterior digestive compartments and survived in bovine feces at 15°C predicting a successful transit of the bacteria along the bovine GIT and its persistence outside the bovine intestine. E. coli MC2 possessed the genetic information encoding 14 adherence systems including adhesins with properties related to colonization of the bovine intestine (F9 fimbriae, EhaA and EspP autotransporters, HCP pilus, FdeC adhesin) reflecting the capacity of the bacteria to colonize different segments of the bovine GIT. E. coli MC2 was also a strong biofilm producer when incubated in fecal samples at low temperature and had a greater ability to form biofilms than the bovine commensal E. coli strain BG1. Furthermore, in contrast to BG1, E. coli MC2 responded to temperature stresses by inducing the genes cspA and htrA during its survival in bovine feces at 15°C. E. coli MC2 also activated genes that are part of the GhoT/GhoS, HicA/HicB and EcnB/EcnA toxin/antitoxin systems involved in the response of E. coli to nutrient starvation and chemical stresses. In summary, the large number of colonization factors known to bind to intestinal epithelium and to biotic or abiotic surfaces, the capacity to produce biofilms and to activate stress fitness genes in bovine feces could explain the persistence of E. coli MC2 in the farm environment.