Gut microbiome biogeography in reindeer supersedes millennia of ecological and evolutionary separation.

Ruminants are dependent on their gut microbiomes for nutrient extraction from plant diets. However, knowledge about the composition, diversity, function, and spatial structure of gut microbiomes, especially in wild ruminants, is limited, largely because analysis has been restricted to faeces or the rumen. In two geographically separated reindeer subspecies, 16S rRNA gene amplicon sequencing revealed strong spatial structuring, and pronounced differences in microbial diversity of at least 33 phyla across the stomach, small intestine, and large intestine (including faeces). The main structural feature was the Bacteroidota to Firmicutes ratio, which declined from the stomach to the large intestine, likely reflecting functional adaptation. Metagenome shotgun sequencing also revealed highly significant structuring in the relative occurrence of carbohydrate-active enzymes (CAZymes). CAZymes were enriched in the rumen relative to the small and large intestines. Interestingly, taxonomic diversity was highest in the large intestine, suggesting an important and understudied role for this organ. Despite the two study populations being separated by an ocean and six millennia of evolutionary history, gut microbiome structuring was remarkably consistent. Our study suggests a strong selection for gut microbiome biogeography along the gastrointestinal tract in reindeer subspecies.

Changes in the fecal microbiota in dogs with acute hemorrhagic diarrhea during an outbreak in Norway.

BACKGROUND: A severe form of acute hemorrhagic diarrhea syndrome (AHDS) occurred in dogs in the Oslo region of Norway during autumn 2019. OBJECTIVES: To characterize the fecal microbiota of dogs with AHDS during the outbreak and compare it to that of healthy dogs from the same period and before the outbreak. ANIMALS: Dogs with AHDS (n = 50), dogs with nonhemorrhagic diarrhea (n = 3), and healthy dogs (n = 11) were sampled during the outbreak. In addition, 78 healthy dogs from the same region were sampled before the outbreak between 2017 and 2018. METHODS: Retrospective case-control study. The fecal microbiotas were characterized using 16S rRNA gene amplicon sequencing. RESULTS: Dogs with AHDS had significantly different microbiota composition (R2  = .07, P < .001) and decreased intestinal diversity relative to healthy dogs from the outbreak period (median, 2.7; range, 0.9-3.5 vs median, 3.2; range, 2.6-4.0; P < .001). The microbiota in dogs with AHDS was characterized by a decrease of Firmicutes and an outgrowth of Proteobacteria, with increased numbers of Clostridium perfringens and Providencia spp. Among the Providencia spp., 1 showed 100% sequence identity with a Providencia alcalifaciens strain that was cultivated and isolated from the same outbreak. No Providencia spp. was found in healthy dogs sampled before the outbreak. CONCLUSIONS AND CLINICAL IMPORTANCE: Dogs with AHDS had marked changes in fecal microbiota including increased numbers of Providencia spp. and C. perfringens, which may have contributed to the severity of this illness.

Individuality and convergence of the infant gut microbiota during the first year of life.

The human gut microbiota plays a vital role in health and disease, and microbial colonization is a key process in infant development. Here, we analyze 2684 fecal specimens from 12 infants during their first year of life, providing detailed insights into the human gut colonization process. Maturation of the gut microbial community shows strong temporal structure and specific developmental stages. At 2-4 months of age, there is a period of accelerated convergence concurrent with a bloom of Bifidobacterium, a genus associated with metabolism of oligosaccharides found in breast milk. The end of this period coincides with the introduction of solid food, a reduction in the relative abundance of Bifidobacterium, and an increase in several groups of Firmicutes. Our findings highlight the dynamic nature and individuality of the gut colonization process, and the need for high-frequency sampling over an extended period when designing and interpreting infant microbiome studies.

Multilevel social structure and diet shape the gut microbiota of the gelada monkey, the only grazing primate.

BACKGROUND: The gelada monkey (Theropithecus gelada), endemic to the Ethiopian highlands, is the only graminivorous primate, i.e., it feeds mainly on grasses and sedges. In spite of known dental, manual, and locomotor adaptations, the intestinal anatomy of geladas is similar to that of other primates. We currently lack a clear understanding of the adaptations in digestive physiology necessary for this species to subsist on a graminoid-based diet, but digestion in other graminivores, such as ruminants, relies heavily on the microbial community residing in the gastrointestinal (GI) system. Furthermore, geladas form complex, multilevel societies, making them a suitable system for investigating links between sociality and the GI microbiota. RESULTS: Here, we explore the gastrointestinal microbiota of gelada monkeys inhabiting an intact ecosystem and document how factors like multilevel social structure and seasonal changes in diet shape the GI microbiota. We compare the gelada GI microbiota to those of other primate species, reporting a gradient from geladas to herbivorous specialist monkeys to dietary generalist monkeys and lastly humans, the ultimate ecological generalists. We also compare the microbiotas of the gelada GI tract and the sheep rumen, finding that geladas are highly enriched for cellulolytic bacteria associated with ruminant digestion, relative to other primates. CONCLUSIONS: This study represents the first analysis of the gelada GI microbiota, providing insights into the adaptations underlying graminivory in a primate. Our results also highlight the role of social organization in structuring the GI microbiota within a society of wild animals.

Ecological plasticity in the gastrointestinal microbiomes of Ethiopian Chlorocebus monkeys.

Human activities can cause habitat degradation that may alter the types and quality of available food resources and thus influence the microbiomes of wild animal populations. Furthermore, seasonal shifts in food availability may cause adaptive responses in the gut microbiome to meet the need for different metabolic capabilities. Here, we demonstrate local-scale population structure in the gastrointestinal microbiotas of Chlorocebus monkeys, in southern Ethiopia, in response to varying degrees of human encroachment. We further provide evidence of adaptation to ecological conditions associated with the dry and wet seasons, and show seasonal effects to be more pronounced in areas with limited human activity. Finally, we report species-level microbiota differences between the endemic Ethiopian Bale monkey, an ecological specialist, and generalist Chlorocebus species from the same geographical region.

Linking Spatial Structure and Community-Level Biotic Interactions through Cooccurrence and Time Series Modeling of the Human Intestinal Microbiota.

The gastrointestinal (GI) microbiome is a densely populated ecosystem where dynamics are determined by interactions between microbial community members, as well as host factors. The spatial organization of this system is thought to be important in human health, yet this aspect of our resident microbiome is still poorly understood. In this study, we report significant spatial structure of the GI microbiota, and we identify general categories of spatial patterning in the distribution of microbial taxa along a healthy human GI tract. We further estimate the biotic interaction structure in the GI microbiota, both through time series and cooccurrence modeling of microbial community data derived from a large number of sequentially collected fecal samples. Comparison of these two approaches showed that species pairs involved in significant negative interactions had strong positive contemporaneous correlations and vice versa, while for species pairs without significant interactions, contemporaneous correlations were distributed around zero. We observed similar patterns when comparing these models to the spatial correlations between taxa identified in the adherent microbiota. This suggests that colocalization of microbial taxon pairs, and thus the spatial organization of the GI microbiota, is driven, at least in part, by direct or indirect biotic interactions. Thus, our study can provide a basis for an ecological interpretation of the biogeography of the human gut. IMPORTANCE The human gut microbiome is the subject of intense study due to its importance in health and disease. The majority of these studies have been based on the analysis of feces. However, little is known about how the microbial composition in fecal samples relates to the spatial distribution of microbial taxa along the gastrointestinal tract. By characterizing the microbial content both in intestinal tissue samples and in fecal samples obtained daily, we provide a conceptual framework for how the spatial structure relates to biotic interactions on the community level. We further describe general categories of spatial distribution patterns and identify taxa conforming to these categories. To our knowledge, this is the first study combining spatial and temporal analyses of the human gut microbiome. This type of analysis can be used for identifying candidate probiotics and designing strategies for clinical intervention.

A novel ultra high-throughput 16S rRNA gene amplicon sequencing library preparation method for the Illumina HiSeq platform.

BACKGROUND: Advances in sequencing technologies and bioinformatics have made the analysis of microbial communities almost routine. Nonetheless, the need remains to improve on the techniques used for gathering such data, including increasing throughput while lowering cost and benchmarking the techniques so that potential sources of bias can be better characterized. METHODS: We present a triple-index amplicon sequencing strategy to sequence large numbers of samples at significantly lower c ost and in a shorter timeframe compared to existing methods. The design employs a two-stage PCR protocol, incorpo rating three barcodes to each sample, with the possibility to add a fourth-index. It also includes heterogeneity spacers to overcome low complexity issues faced when sequencing amplicons on Illumina platforms. RESULTS: The library preparation method was extensively benchmarked through analysis of a mock community in order to assess biases introduced by sample indexing, number of PCR cycles, and template concentration. We further evaluated the method through re-sequencing of a standardized environmental sample. Finally, we evaluated our protocol on a set of fecal samples from a small cohort of healthy adults, demonstrating good performance in a realistic experimental setting. Between-sample variation was mainly related to batch effects, such as DNA extraction, while sample indexing was also a significant source of bias. PCR cycle number strongly influenced chimera formation and affected relative abundance estimates of species with high GC content. Libraries were sequenced using the Illumina HiSeq and MiSeq platforms to demonstrate that this protocol is highly scalable to sequence thousands of samples at a very low cost. CONCLUSIONS: Here, we provide the most comprehensive study of performance and bias inherent to a 16S rRNA gene amplicon sequencing method to date. Triple-indexing greatly reduces the number of long custom DNA oligos required for library preparation, while the inclusion of variable length heterogeneity spacers minimizes the need for PhiX spike-in. This design results in a significant cost reduction of highly multiplexed amplicon sequencing. The biases we characterize highlight the need for highly standardized protocols. Reassuringly, we find that the biological signal is a far stronger structuring factor than the various sources of bias.

Saffold Virus, a Human Cardiovirus, and Risk of Persistent Islet Autoantibodies in the Longitudinal Birth Cohort Study MIDIA.

The aim of this study was to describe the frequency and distribution of Saffold virus in longitudinal stool samples from children, and test for association with development of persistent autoantibodies predictive of type 1 diabetes. A cohort of Norwegian children carrying the HLA genotype associated with highest risk of type 1 diabetes ("DR4-DQ8/DR3-DQ2") was followed with monthly stool samples from 3 to 35 months of age. Blood samples were tested for autoantibodies to insulin, glutamic acid decarboxylase65 and Islet Antigen-2. 2077 stool samples from 27 children with ≥ 2 repeatedly positive islet autoantibodies (cases), and 53 matched controls were analysed for Saffold virus genomic RNA by semi-quantitative real-time reverse transcriptase PCR. Saffold virus was found in 53 of 2077 (2.6%) samples, with similar proportions between cases (2.5%) and controls (2.6%). The probability of being infected by 3 years of age was 28% (95% CI 0.18-0.40). Viral quantities ranged from <1 to almost 105 copies/µl. Estimated odds ratio between islet autoimmunity and infection episodes prior to seroconversion was 1.98 (95% CI: 0.57-6.91, p = 0.29). Saffold virus had no statistically significant association with islet autoimmunity.

Context-dependent competition in a model gut bacterial community.

Understanding the ecological processes that generate complex community structures may provide insight into the establishment and maintenance of a normal microbial community in the human gastrointestinal tract, yet very little is known about how biotic interactions influence community dynamics in this system. Here, we use natural strains of Escherichia coli and a simplified model microbiota to demonstrate that the colonization process on the strain level can be context dependent, in the sense that the outcome of intra-specific competition may be determined by the composition of the background community. These results are consistent with previous models for competition between organisms where one competitor has adapted to low resource environments whereas the other is optimized for rapid reproduction when resources are abundant. The genomic profiles of E. coli strains representing these differing ecological strategies provide clues for deciphering the genetic underpinnings of niche adaptation within a single species. Our findings extend the role of ecological theory in understanding microbial systems and the conceptual toolbox for describing microbial community dynamics. There are few, if any, concrete examples of context-dependent competition on a single trophic level. However, this phenomenon can have potentially dramatic effects on which bacteria will successfully establish and persist in the gastrointestinal system, and the principle should be equally applicable to other microbial ecosystems.

Comparisons of infant Escherichia coli isolates link genomic profiles with adaptation to the ecological niche.

BACKGROUND: Despite being one of the most intensely studied model organisms, many questions still remain about the evolutionary biology and ecology of Escherichia coli. An important step toward achieving a more complete understanding of E.coli biology entails elucidating relationships between gene content and adaptation to the ecological niche. RESULTS: Here, we present genome comparisons of 16 E.coli strains that represent commensals and pathogens isolated from infants during a specific time period in Trondheim, Norway. Using differential gene content, we characterized enrichment profiles of the collection of strains relating to phylogeny, early vs. late colonization, pathogenicity and growth rate. We found clear gene content distinctions relating to the various grouping criteria. We also found that different categories of strains use different genetic elements for similar biological processes. The sequenced genomes included two pairs of strains where each pair was isolated from the same infant at different time points. One pair, in which the strains were isolated four months apart, showed maintenance of an early colonizer genome profile but also gene content and codon usage changes toward the late colonizer profile. Lastly, we placed our sequenced isolates into a broader genomic context by comparing them with 25 published E.coli genomes that represent a variety of pathotypes and commensal strains. This analysis demonstrated the importance of geography in shaping strain level gene content profiles. CONCLUSIONS: Our results indicate a general pattern where alternative genetic pathways lead toward a consistent ecological role for E.coli as a species. Within this framework however, we saw selection shaping the coding repertoire of E.coli strains toward distinct ecotypes with different phenotypic properties.

Diversity, transmission and persistence of Escherichia coli in a cohort of mothers and their infants.

Despite that Escherichia coli is one of the most important bacteria in early infant colonization and immune modulation, we have limited knowledge about diversity, transmission and persistence within human populations for this bacterium. Here we have utilized a novel, growth-independent, direct typing approach to describe E. coli mother-to-child transmission and persistence within infants in a well-defined cohort of 86 mothers and their infants in Norway. Seven gene multilocus sequence typing of 28 study isolates, three probiotic strains, eight Norwegian pathogenic isolates and the ECOR strain collection added a phylogenetic framework to the direct sequencing data. We found that a type B2 subpopulation of the maternal E. coli strains was the main group transmitted to the infants and that the proportion of children carrying their mothers' strain decreased as the children age. Using species richness estimates we also found a limited number of strains within the cohort compared with total E. coli diversity, constraints on infant colonization, and that infant strain diversity levels increased towards maternal diversity levels over time. This knowledge about inheritance and diversity forms a foundation for future understanding of E. coli in human health and disease.

N-Glycan Moieties in Neonatal Fc Receptor Determine Steady-state Membrane Distribution and Directional Transport of IgG.

The neonatal Fc receptor (FcRn) is a major histocompatibility complex class I-related molecule known to protect IgG and albumin from catabolism and transport IgG across polarized epithelial cells in a bidirectional manner. Previous studies have shown species-specific differences in ligand binding, IgG transport direction, and steady-state membrane distribution when expressed in polarized epithelial cells. We hypothesized that these differences may be due to the additional N-glycans expressed on the rat FcRn, because N-glycans have been proposed to function as apical targeting signals, and that two of the N-glycan moieties have been shown to contribute to the IgG binding of rat FcRn. A panel of mutant human FcRn variants was generated to resemble the N-glycan expression of rat FcRn in various combinations and subsequently transfected into Madin-Darby canine kidney II cells together with human beta2-microglobulin. Mutant human FcRn clones that contained additional N-glycan side-chain modifications, including that which was fully rodentized, still exhibited specificity for human IgG and failed to bind to mouse IgG. At steady state, the mutant human FcRn with additional N-glycans redistributed to the apical cell surface similar to that of rat FcRn. Furthermore, the rodentized human FcRn exhibited a reversal of IgG transport with predominant transcytosis from an apical-to-basolateral direction, which resembled that of the rat FcRn isoform. These studies show that the N-glycans in FcRn contribute significantly to the steady-state membrane distribution and direction of IgG transport in polarized epithelia.