Impacts of sample handling and storage conditions on archiving physiologically active soil microbial communities.

Soil microbial communities are fundamental to ecosystem processes and plant growth, yet community composition is seasonally and successionally dynamic, which interferes with long-term iterative experimentation of plant-microbe interactions. We explore how soil sample handling (e.g. filtering) and sample storage conditions impact the ability to revive the original, physiologically active, soil microbial community. We obtained soil from agricultural fields in Montana and Oklahoma, USA and samples were sieved to 2 mm or filtered to 45 µm. Sieved and filtered soil samples were archived at -20°C or -80°C for 50 days and revived for 2 or 7 days. We extracted DNA and the more transient RNA pools from control and treatment samples and characterized microbial communities using 16S amplicon sequencing. Filtration and storage treatments significantly altered soil microbial communities, impacting both species richness and community composition. Storing sieved soil at -20°C did not alter species richness and resulted in the least disruption to the microbial community composition in comparison to nonarchived controls as characterized by RNA pools from soils of both sites. Filtration significantly altered composition but not species richness. Archiving sieved soil at -20°C could allow for long-term and repeated experimentation on preserved physiologically active microbial communities.

A novel conjugative transposon carrying an autonomously amplified plasmid.

Tetracyclines serve as broad-spectrum antibiotics to treat bacterial infections. The discovery of new tetracycline resistance genes has led to new questions about the underlying mechanisms of resistance, gene transfer, and their relevance to human health. We tracked changes in the abundance of a 55-kbp conjugative transposon (CTn214) carrying tetQ, a tetracycline resistance gene, within a Bacteroides fragilis metagenome-assembled genome derived from shotgun sequencing of microbial DNA extracted from the ileal pouch of a patient with ulcerative colitis. The mapping of metagenomic reads to CTn214 revealed the multi-copy nature of a 17,044-nt region containing tetQ in samples collected during inflammation and uninflamed visits. B. fragilis cultivars isolated from the same patient during periods of inflammation harbored CTn214 integrated into the chromosome or both a circular, multi-copy, extrachromosomal region of the CTn214 containing tetQ and the corresponding integrated form. The tetracycline-dependent mechanism for the transmission of CTn214 is nearly identical to a common conjugative transposon found in the genome of B. fragilis (CTnDOT), but the autonomously amplified nature of a circular 17,044-nt region of CTn214 that codes for tetQ and the integration of the same sequence in the linear chromosome within the same cell is a novel observation. Genome and transcriptome sequencing of B. fragilis cultivars grown under different concentrations of tetracycline and ciprofloxacin indicates that tetQ in strains containing the circular form remains actively expressed regardless of treatment, while the expression of tetQ in strains containing the linear form increases only in the presence of tetracycline.IMPORTANCEThe exchange of antibiotic production and resistance genes between microorganisms can lead to the emergence of new pathogens. In this study, short-read mapping of metagenomic samples taken over time from the illeal pouch of a patient with ulcerative colitis to a Bacteroides fragilis metagenome-assembled genome revealed two distinct genomic arrangements of a novel conjugative transposon, CTn214, that encodes tetracycline resistance. The autonomous amplification of a plasmid-like circular form from CTn214 that includes tetQ potentially provides consistent ribosome protection against tetracycline. This mode of antibiotic resistance offers a novel mechanism for understanding the emergence of pathobionts like B. fragilis and their persistence for extended periods of time in patients with inflammatory bowel disease.

Human milk-associated bacterial communities associate with the infant gut microbiome over the first year of life.

Introduction: Microbial communities inhabiting the human infant gut are important for immune system development and lifelong health. One critical exposure affecting the bacterial colonization of the infant gut is consumption of human milk, which contains diverse microbial communities and prebiotics. We hypothesized that human milk-associated microbial profiles are associated with those of the infant gut. Methods: Maternal-infant dyads enrolled in the New Hampshire Birth Cohort Study (n = 189 dyads) contributed breast milk and infant stool samples collected approximately at 6 weeks, 4 months, 6 months, 9 months, and 12 months postpartum (n = 572 samples). Microbial DNA was extracted from milk and stool and the V4-V5 region of the bacterial 16S rRNA gene was sequenced. Results: Clustering analysis identified three breast milk microbiome types (BMTs), characterized by differences in Streptococcus, Staphylococcus, Pseudomonas, Acinetobacter, and microbial diversity. Four 6-week infant gut microbiome types (6wIGMTs) were identified, differing in abundances of Bifidobacterium, Bacteroides, Clostridium, Streptococcus, and Escherichia/Shigella, while two 12-month IGMTs (12mIGMTs) differed primarily by Bacteroides presence. At 6 weeks, BMT was associated with 6wIGMT (Fisher's exact test value of p = 0.039); this association was strongest among infants delivered by Cesarean section (Fisher's exact test value of p = 0.0028). The strongest correlations between overall breast milk and infant stool microbial community structures were observed when comparing breast milk samples to infant stool samples collected at a subsequent time point, e.g., the 6-week breast milk microbiome associated with the 6-month infant gut microbiome (Mantel test Z-statistic = 0.53, value of p = 0.001). Streptoccous and Veillonella species abundance were correlated in 6-week milk and infant stool, and 4- and 6-month milk Pantoea species were associated with infant stool Lachnospiraceae genera at 9 and 12 months. Discussion: We identified clusters of human milk and infant stool microbial communities that were associated in maternal-infant dyads at 6 weeks of life and found that milk microbial communities were more strongly associated with infant gut microbial communities in infants delivered operatively and after a lag period. These results suggest that milk microbial communities have a long-term effect on the infant gut microbiome both through sharing of microbes and other molecular mechanisms.

Metabolic independence drives gut microbial colonization and resilience in health and disease.

BACKGROUND: Changes in microbial community composition as a function of human health and disease states have sparked remarkable interest in the human gut microbiome. However, establishing reproducible insights into the determinants of microbial succession in disease has been a formidable challenge. RESULTS: Here we use fecal microbiota transplantation (FMT) as an in natura experimental model to investigate the association between metabolic independence and resilience in stressed gut environments. Our genome-resolved metagenomics survey suggests that FMT serves as an environmental filter that favors populations with higher metabolic independence, the genomes of which encode complete metabolic modules to synthesize critical metabolites, including amino acids, nucleotides, and vitamins. Interestingly, we observe higher completion of the same biosynthetic pathways in microbes enriched in IBD patients. CONCLUSIONS: These observations suggest a general mechanism that underlies changes in diversity in perturbed gut environments and reveal taxon-independent markers of "dysbiosis" that may explain why widespread yet typically low-abundance members of healthy gut microbiomes can dominate under inflammatory conditions without any causal association with disease.

The rhizosphere microbiome and host plant glucosinolates exhibit feedback cycles in Brassica rapa.

The rhizosphere microbiome influences many aspects of plant fitness, including production of secondary compounds and defence against insect herbivores. Plants also modulate the composition of the microbial community in the rhizosphere via secretion of root exudates. We tested both the effect of the rhizosphere microbiome on plant traits, and host plant effects on rhizosphere microbes using recombinant inbred lines (RILs) of Brassica rapa that differ in production of glucosinolates (GLS), secondary metabolites that contribute to defence against insect herbivores. First, we investigated the effect of genetic variation in GLS production on the composition of the rhizosphere microbiome. Using a Bayesian Dirichlet-multinomial regression model (DMBVS), we identified both negative and positive associations between bacteria from six genera and the concentration of five GLS compounds produced in plant roots. Additionally, we tested the effects of microbial inoculation (an intact vs. disrupted soil microbiome) on GLS production and insect damage in these RILs. We found a significant microbial treatment × genotype interaction, in which total GLS was higher in the intact relative to the disrupted microbiome treatment in some RILs. However, despite differences in GLS production between microbial treatments, we observed no difference in insect damage between treatments. Together, these results provide evidence for a full feedback cycle of plant-microbe interactions mediated by GLS; that is, GLS compounds produced by the host plant "feed-down" to influence rhizosphere microbial community and rhizosphere microbes "feed-up" to influence GLS production.

A prospective study of the infant gut microbiome in relation to vaccine response.

BACKGROUND: The establishment of the gut microbiome plays a key symbiotic role in the developing immune system; however, its influence on vaccine response is yet uncertain. We prospectively investigated the composition and diversity of the early-life gut microbiome in relation to infant antibody response to two routinely administered vaccines. METHODS: Eighty-three infants enrolled in the New Hampshire Birth Cohort Study were included in the analysis. We collected blood samples at 12 months of age and assayed the isolated serum to quantify total IgG and measured antibody to pneumococcal capsular polysaccharide and tetanus toxoid. Stool samples were collected from infants at 6 weeks of age and sequenced using 16S rRNA, and a subset of 61 samples were sequenced using shotgun metagenomics sequencing. RESULTS: We observed differences in beta diversity for 16S 6-week stool microbiota and pneumococcal and tetanus IgG antibody responses. Metagenomics analyses identified species and metabolic pathways in 6-week stool associated with tetanus antibody response, in particular, negative associations with the relative abundance of Aeriscardovia aeriphila species and positive associations with the relative abundance of species associated with CDP-diacylglycerol biosynthesis pathways. CONCLUSIONS: The early gut microbiome composition may influence an infant's vaccine response. IMPACT: Early intestinal microbiome acquisition plays a critical role in immune maturation and in both adaptive and innate immune response in infancy. We identified associations between early life microbiome composition and response to two routinely administered vaccines-pneumococcal capsular polysaccharide and tetanus toxoid-measured at approximately 1 year of age. Our findings highlight the potential impact of the gut microbiome on infant immune response that may open up opportunities for future interventions.

The relationship between the gut microbiome and the risk of respiratory infections among newborns.

Background: Emerging evidence points to a critical role of the developing gut microbiome in immune maturation and infant health; however, prospective studies are lacking. Methods: We examined the occurrence of infections and associated symptoms during the first year of life in relation to the infant gut microbiome at six weeks of age using bacterial 16S rRNA V4-V5 gene sequencing (N = 465) and shotgun metagenomics (N = 185). We used generalized estimating equations to assess the associations between longitudinal outcomes and 16S alpha diversity and metagenomics species. Results: Here we show higher infant gut microbiota alpha diversity was associated with an increased risk of infections or respiratory symptoms treated with a prescription medicine, and specifically upper respiratory tract infections. Among vaginally delivered infants, a higher alpha diversity was associated with an increased risk of all-cause wheezing treated with a prescription medicine and diarrhea involving a visit to a health care provider. Positive associations were specifically observed with Veillonella species among all deliveries and Haemophilus influenzae among cesarean-delivered infants. Conclusion: Our findings suggest that intestinal microbial diversity and the relative abundance of key taxa in early infancy may influence susceptibility to respiratory infection, wheezing, and diarrhea.

Geochemical and microbiological profiles in hydrothermal extreme acidic environments (Pisciarelli Spring, Campi Flegrei, Italy).

Although terrestrial hydrothermal systems are considered among the most fascinating environments, how their unique and extreme conditions can affect microorganisms selection and the role in biogeochemical cycles has not yet been well elucidated. A combined geochemical and microbiological exploration in waters and sediments from 10 sampling points along a sharp temperature gradient (15-90°C) within an extremely acidic hydrothermal system (Pisciarelli Spring, Campi Flegrei area, southern Italy) displayed how hydrothermal fluids influence the microbial dynamics. This area was characterized by high levels of reduced gaseous species (e.g. H2S, H2, CH4, CO) and very low pH values (<2.3). Thermodynamic calculations revealed a high microbial catabolic potential in oxidation/reduction reactions of N-, S- and Fe-bearing species. Overall, an increase of the archaeal/bacterial abundance ratio was observed by decreasing temperature and pH values. In particular, Archaea and Bacteria were present in almost equal cell abundance (up to 1.1 × 109 and 9.3 × 108 cell/g, respectively) in the <70°C sampling points (average pH = 2.09); on the contrary, the highest temperature waters (85-90°C; average pH = 2.26) were characterized by a low abundance of archaeal cells. The high-throughput sequencing of the 16S rRNA genes indicated strong differences in archaeal and bacterial communities composition along the temperature gradient. However, the microbiome in this extreme environment was mainly constituted by chemoautotrophic microorganisms that were likely involved in N-, S- and Fe-bearing species transformations (e.g. Acidianus infernus, Ferroplasma acidarmanus, Acidithiobacillus,Sulfobacillus,Thaumarchaeota), in agreement with thermodynamic calculations.

Impact of antibiotics on off-target infant gut microbiota and resistance genes in cohort studies.

BACKGROUND: Young children are frequently exposed to antibiotics, with the potential for collateral consequences to the gut microbiome. The impact of antibiotic exposures to off-target microbes (i.e., bacteria not targeted by treatment) and antibiotic resistance genes (ARGs) is poorly understood. METHODS: We used metagenomic sequencing data from paired stool samples collected prior to antibiotic exposure and at 1 year from over 200 infants and a difference-in-differences approach to assess the relationship between subsequent exposures and the abundance or compositional diversity of microbes and ARGs while adjusting for covariates. RESULTS: By 1 year, the abundance of multiple species and ARGs differed by antibiotic exposure. Compared to infants never exposed to antibiotics, Bacteroides vulgatus relative abundance increased by 1.72% (95% CI: 0.19, 3.24) while Bacteroides fragilis decreased by 1.56% (95% CI: -4.32, 1.21). Bifidobacterium species also exhibited opposing trends. ARGs associated with exposure included class A beta-lactamase gene CfxA6. Among infants attending day care, Escherichia coli and ARG abundance were both positively associated with antibiotic use. CONCLUSION: Novel findings, including the importance of day care attendance, were identified through considering microbiome data at baseline and post-intervention. Thus, our study design and approach have important implications for future studies evaluating the unintended impacts of antibiotics. IMPACT: The impact of antibiotic exposure to off-target microbes and antibiotic resistance genes in the gut is poorly defined. We quantified these impacts in two cohort studies using a difference-in-differences approach. Novel to microbiome studies, we used pre/post-antibiotic data to emulate a randomized controlled trial. Compared to infants unexposed to antibiotics between baseline and 1 year, the relative abundance of multiple off-target species and antibiotic resistance genes was altered. Infants who attended day care and were exposed to antibiotics within the first year had a higher abundance of Escherichia coli and antibiotic resistance genes; a novel finding warranting further investigation.

Spatiotemporal Heterogeneity and Intragenus Variability in Rhizobacterial Associations with Brassica rapa Growth.

Microbial communities in the rhizosphere are distinct from those in soils and are influenced by stochastic and deterministic processes during plant development. These communities contain bacteria capable of promoting growth in host plants through various strategies. While some interactions are characterized in mechanistic detail using model systems, others can be inferred from culture-independent methods, such as 16S amplicon sequencing, using machine learning methods that account for this compositional data type. To characterize assembly processes and identify community members associated with plant growth amid the spatiotemporal variability of the rhizosphere, we grew Brassica rapa in a greenhouse time series with amended and reduced microbial treatments. Inoculation with a native soil community increased plant leaf area throughout the time series by up to 28%. Despite identifying spatially and temporally variable amplicon sequence variants (ASVs) in both treatments, inoculated communities were more highly connected and assembled more deterministically overall. Using a generalized linear modeling approach controlling for spatial variability, we identified 43 unique ASVs that were positively or negatively associated with leaf area, biomass, or growth rates across treatments and time stages. ASVs of the genus Flavobacterium dominated rhizosphere communities and showed some of the strongest positive and negative correlations with plant growth. Members of this genus, and growth-associated ASVs more broadly, exhibited variable connectivity in networks independent of growth association (positive or negative). These findings suggest host-rhizobacterial interactions vary temporally at narrow taxonomic scales and present a framework for identifying rhizobacteria that may work independently or in concert to improve agricultural yields. IMPORTANCE The rhizosphere, the zone of soil surrounding plant roots, is a hot spot for microbial activity, hosting bacteria capable of promoting plant growth in ways like increasing nutrient availability or fighting plant pathogens. This microbial system is highly diverse and most bacteria are unculturable, so to identify specific bacteria associated with plant growth, we used culture-independent community DNA sequencing combined with machine learning techniques. We identified 43 specific bacterial sequences associated with the growth of the plant Brassica rapa in different soil microbial treatments and at different stages of plant development. Most associations between bacterial abundances and plant growth were positive, although similar bacterial groups sometimes had different effects on growth. Why this happens will require more research, but overall, this study provides a way to identify native bacteria from plant roots that might be isolated and applied to boost agricultural yields.

High molecular weight DNA extraction strategies for long-read sequencing of complex metagenomes.

By offering extremely long contiguous characterization of individual DNA molecules, rapidly emerging long-read sequencing strategies offer comprehensive insights into the organization of genetic information in genomes and metagenomes. However, successful long-read sequencing experiments demand high concentrations of highly purified DNA of high molecular weight (HMW), which limits the utility of established DNA extraction kits designed for short-read sequencing. The challenges associated with input DNA quality intensify further when working with complex environmental samples of low microbial biomass, which requires new protocols that are tailored to study metagenomes with long-read sequencing. Here, we use human tongue scrapings to benchmark six HMW DNA extraction strategies that are based on commercially available kits, phenol-chloroform (PC) extraction and agarose encasement followed by agarase digestion. A typical end goal of HMW DNA extractions is to obtain the longest possible reads during sequencing, which is often achieved by PC extractions, as demonstrated in sequencing of cultured cells. Yet our analyses that consider overall read-size distribution, assembly performance and the number of circularized elements found in sequencing results suggest that column-based kits with enzyme supplementation, rather than PC methods, may be more appropriate for long-read sequencing of metagenomes.

Associations between the gut microbiome and metabolome in early life.

BACKGROUND: The infant intestinal microbiome plays an important role in metabolism and immune development with impacts on lifelong health. The linkage between the taxonomic composition of the microbiome and its metabolic phenotype is undefined and complicated by redundancies in the taxon-function relationship within microbial communities. To inform a more mechanistic understanding of the relationship between the microbiome and health, we performed an integrative statistical and machine learning-based analysis of microbe taxonomic structure and metabolic function in order to characterize the taxa-function relationship in early life. RESULTS: Stool samples collected from infants enrolled in the New Hampshire Birth Cohort Study (NHBCS) at approximately 6-weeks (n = 158) and 12-months (n = 282) of age were profiled using targeted and untargeted nuclear magnetic resonance (NMR) spectroscopy as well as DNA sequencing of the V4-V5 hypervariable region from the bacterial 16S rRNA gene. There was significant inter-omic concordance based on Procrustes analysis (6 weeks: p = 0.056; 12 months: p = 0.001), however this association was no longer significant when accounting for phylogenetic relationships using generalized UniFrac distance metric (6 weeks: p = 0.376; 12 months: p = 0.069). Sparse canonical correlation analysis showed significant correlation, as well as identifying sets of microbe/metabolites driving microbiome-metabolome relatedness. Performance of machine learning models varied across different metabolites, with support vector machines (radial basis function kernel) being the consistently top ranked model. However, predictive R2 values demonstrated poor predictive performance across all models assessed (avg: - 5.06% -- 6 weeks; - 3.7% -- 12 months). Conversely, the Spearman correlation metric was higher (avg: 0.344-6 weeks; 0.265-12 months). This demonstrated that taxonomic relative abundance was not predictive of metabolite concentrations. CONCLUSIONS: Our results suggest a degree of overall association between taxonomic profiles and metabolite concentrations. However, lack of predictive capacity for stool metabolic signatures reflects, in part, the possible role of functional redundancy in defining the taxa-function relationship in early life as well as the bidirectional nature of the microbiome-metabolome association. Our results provide evidence in favor of a multi-omic approach for microbiome studies, especially those focused on health outcomes.

The infant gut resistome is associated with E. coli and early-life exposures.

BACKGROUND: The human gut microbiome harbors a collection of bacterial antimicrobial resistance genes (ARGs) known as the resistome. The factors associated with establishment of the resistome in early life are not well understood. We investigated the early-life exposures and taxonomic signatures associated with resistome development over the first year of life in a large, prospective cohort in the United States. Shotgun metagenomic sequencing was used to profile both microbial composition and ARGs in stool samples collected at 6 weeks and 1 year of age from infants enrolled in the New Hampshire Birth Cohort Study. Negative binomial regression and statistical modeling were used to examine infant factors such as sex, delivery mode, feeding method, gestational age, antibiotic exposure, and infant gut microbiome composition in relation to the diversity and relative abundance of ARGs. RESULTS: Metagenomic sequencing was performed on paired samples from 195 full term (at least 37 weeks' gestation) and 15 late preterm (33-36 weeks' gestation) infants. 6-week samples compared to 1-year samples had 4.37 times (95% CI: 3.54-5.39) the rate of harboring ARGs. The majority of ARGs that were at a greater relative abundance at 6 weeks (chi-squared p < 0.01) worked through the mechanism of antibiotic efflux. The overall relative abundance of the resistome was strongly correlated with Proteobacteria (Spearman correlation = 78.9%) and specifically Escherichia coli (62.2%) relative abundance in the gut microbiome. Among infant characteristics, delivery mode was most strongly associated with the diversity and relative abundance of ARGs. Infants born via cesarean delivery had a trend towards a higher risk of harboring unique ARGs [relative risk = 1.12 (95% CI: 0.97-1.29)] as well as having an increased risk for overall ARG relative abundance [relative risk = 1.43 (95% CI: 1.12-1.84)] at 1 year compared to infants born vaginally. CONCLUSIONS: Our findings suggest that the developing infant gut resistome may be alterable by early-life exposures. Establishing the extent to which infant characteristics and early-life exposures impact the resistome can ultimately lead to interventions that decrease the transmission of ARGs and thus the risk of antibiotic resistant infections.

Time outweighs the effect of host developmental stage on microbial community composition.

Thousands of microbial taxa in the soil form symbioses with host plants, and due to their contribution to plant performance, these microbes are often considered an extension of the host genome. Given microbial effects on host performance, it is important to understand factors that govern microbial community assembly. Host developmental stage could affect rhizosphere microbial diversity while, alternatively, microbial assemblages could change simply as a consequence of time and the opportunity for microbial succession. Previous studies suggest that rhizosphere microbial assemblages shift across plant developmental stages, but time since germination is confounded with developmental stage. We asked how elapsed time and potential microbial succession relative to host development affected microbial diversity in the rhizosphere using monogenic flowering-time mutants of Arabidopsis thaliana. Under our experimental design, different developmental stages were present among host genotypes after the same amount of time following germination, e.g. at 76 days following germination some host genotypes were flowering while others were fruiting or senescing. We found that elapsed time was a strong predictor of microbial diversity whereas there were few differences among developmental stages. Our results support the idea that time and, likely, microbial succession more strongly affect microbial community assembly than host developmental stage.

The microbiome of the habitat-forming brown alga Fucus vesiculosus (Phaeophyceae) has similar cross-Atlantic structure that reflects past and present drivers1.

Latitudinal diversity gradients have provided many insights into species differentiation and community processes. In the well-studied intertidal zone, however, little is known about latitudinal diversity in microbiomes associated with habitat-forming hosts. We investigated microbiomes of Fucus vesiculosus because of deep understanding of this model system and its latitudinally large, cross-Atlantic range. Given multiple effects of photoperiod, we predicted that cross-Atlantic microbiomes of the Fucus microbiome would be similar at similar latitudes and correlate with environmental factors. We found that community structure and individual amplicon sequencing variants (ASVs) showed distinctive latitudinal distributions, but alpha diversity did not. Latitudinal differentiation was mostly driven by ASVs that were more abundant in cold temperate to subarctic (e.g., Granulosicoccus_t3260, Burkholderia/Caballeronia/Paraburkholderia_t8371) or warm temperate (Pleurocapsa_t10392) latitudes. Their latitudinal distributions correlated with different humidity, tidal heights, and air/sea temperatures, but rarely with irradiance or photoperiod. Many ASVs in potentially symbiotic genera displayed novel phylogenetic biodiversity with differential distributions among tissues and regions, including closely related ASVs with differing north-south distributions that correlated with Fucus phylogeography. An apparent southern range contraction of F. vesiculosus in the NW Atlantic on the North Carolina coast mimics that recently observed in the NE Atlantic. We suggest cross-Atlantic microbial structure of F. vesiculosus is related to a combination of past (glacial-cycle) and contemporary environmental drivers.

Infant Feeding Alters the Longitudinal Impact of Birth Mode on the Development of the Gut Microbiota in the First Year of Life.

Cesarean-delivered (CD) infants harbor a distinct gut microbiome from vaginally delivered (VD) infants, however, during infancy, the most important driver of infant gut microbial colonization is infant feeding. Earlier studies have shown that breastfeeding is associated with higher levels of health-promoting bacteria such and Bifidobacterium and Bacteroides via modulation of the immune system, and production of metabolites. As the infant gut matures and solid foods are introduced, it is unclear whether longer duration of breast feeding restore loss of beneficial taxa within the intestinal microbiota of operatively delivered infants. Within the New Hampshire Birth Cohort Study, we evaluated the longitudinal effect of delivery mode and infant feeding on the taxonomic composition and functional capacity of developing gut microbiota in the First year of life. Microbiota of 500 stool samples collected between 6 weeks and 12 months of age (from 229 infants) were characterized by 16S ribosomal RNA sequencing. Shotgun metagenomic sequencing was also performed on 350 samples collected at either 6 weeks or 12 months of age. Among infant participants, 28% were cesarean-delivered (CD) infants and most (95%) initiated breastfeeding within the first six months of life, with 26% exclusively breastfed and 69% mixed-fed (breast milk and formula), in addition to complementary foods by age 1. Alpha (within-sample) diversity was significantly lower in CD infants compared to vaginally delivered (VD) infants (P < 0.05) throughout the study period. Bacterial community composition clustering by both delivery mode and feeding duration at 1 year of age revealed that CD infants who were breast fed for < 6 months were more dissimilar to VD infants than CD infants who breast fed for ≥ 6 months. We observed that breastfeeding modified the longitudinal impact of delivery mode on the taxonomic composition of the microbiota by 1 year of age, with an observed increase in abundance of Bacteroides fragilis and Lactobacillus with longer duration of breastfeeding among CD infants while there was an increase in Faecalibacterium for VD infants. Our findings confirm that duration of breastfeeding plays a critical role in restoring a health-promoting microbiome, call for further investigations regarding the association between breast milk exposure and health outcomes in early life.

Soil Microsite Outweighs Cultivar Genotype Contribution to Brassica Rhizobacterial Community Structure.

Microorganisms residing on root surfaces play a central role in plant development and performance and may promote growth in agricultural settings. Studies have started to uncover the environmental parameters and host interactions governing their assembly. However, soil microbial communities are extremely diverse and heterogeneous, showing strong variations over short spatial scales. Here, we quantify the relative effect of meter-scale variation in soil bacterial community composition among adjacent field microsites, to better understand how microbial communities vary by host plant genotype as well as soil microsite heterogeneity. We used bacterial 16S rDNA amplicon sequencing to compare rhizosphere communities from four Brassica rapa cultivars grown in three contiguous field plots (blocks) and evaluated the relative contribution of resident soil communities and host genotypes in determining rhizosphere community structure. We characterize concomitant meter-scale variation in bacterial community structure among soils and rhizospheres and show that this block-scale variability surpasses the influence of host genotype in shaping rhizosphere communities. We identified biomarker amplicon sequence variants (ASVs) associated with bulk soil and rhizosphere habitats, each block, and three of four cultivars. Numbers and percent abundances of block-specific biomarkers in rhizosphere communities far surpassed those from bulk soils. These results highlight the importance of fine-scale variation in the pool of colonizing microorganisms during rhizosphere assembly and demonstrate that microsite variation may constitute a confounding effect while testing biotic and abiotic factors governing rhizosphere community structure.

Functional and genetic markers of niche partitioning among enigmatic members of the human oral microbiome.

INTRODUCTION: Microbial residents of the human oral cavity have long been a major focus of microbiology due to their influence on host health and intriguing patterns of site specificity amidst the lack of dispersal limitation. However, the determinants of niche partitioning in this habitat are yet to be fully understood, especially among taxa that belong to recently discovered branches of microbial life. RESULTS: Here, we assemble metagenomes from tongue and dental plaque samples from multiple individuals and reconstruct 790 non-redundant genomes, 43 of which resolve to TM7, a member of the Candidate Phyla Radiation, forming six monophyletic clades that distinctly associate with either plaque or tongue. Both pangenomic and phylogenomic analyses group tongue-specific clades with other host-associated TM7 genomes. In contrast, plaque-specific TM7 group with environmental TM7 genomes. Besides offering deeper insights into the ecology, evolution, and mobilome of cryptic members of the oral microbiome, our study reveals an intriguing resemblance between dental plaque and non-host environments indicated by the TM7 evolution, suggesting that plaque may have served as a stepping stone for environmental microbes to adapt to host environments for some clades of microbes. Additionally, we report that prophages are widespread among oral-associated TM7, while absent from environmental TM7, suggesting that prophages may have played a role in adaptation of TM7 to the host environment. CONCLUSIONS: Our data illuminate niche partitioning of enigmatic members of the oral cavity, including TM7, SR1, and GN02, and provide genomes for poorly characterized yet prevalent members of this biome, such as uncultivated Flavobacteriaceae.

Bacterial Communities Show Algal Host (Fucus spp.)/Zone Differentiation Across the Stress Gradient of the Intertidal Zone.

The intertidal zone often has varying levels of environmental stresses (desiccation, temperature, light) that result in highly stress-tolerant macrobiota occupying the upper zone while less tolerant species occupy the lower zone, but little comparative information is available for intertidal bacteria. Here we describe natural (unmanipulated) bacterial communities of three Fucus congeners (F. spiralis, high zone; F. vesiculosus, mid zone; F. distichus, low zone) as well as those of F. vesiculosus transplanted to the high zone (Dry and Watered treatments) and to the mid zone (Procedural Control) during summer in Maine (United States). We predicted that bacterial communities would be different among the differently zoned natural congeners, and that higher levels of desiccation stress in the high zone would cause bacterial communities of Dry transplants to become similar to F. spiralis, whereas relieving desiccation stress on Watered transplants would maintain the mid-zone F. vesiculosus bacterial community. Bacteria were identified as amplicon sequence variants (ASVs) after sequencing the V4 hypervariable region of the 16S rRNA gene. Microbiome composition and structure were significantly different between the differently zoned congeners at each tissue type (holdfasts, receptacles, vegetative tips). ASVs significantly associated with the mid-zone congener were frequently also present on the high-zone or low-zone congener, whereas overlap in ASVs between the high-zone and low-zone congeners was rare. Only 7 of 6,320 total ASVs were shared among tissues over all congeners and transplant treatments. Holdfast bacterial community composition of Dry transplants was not significantly different from that of F. spiralis, but Watered holdfast communities were significantly different from those of F. spiralis and not significantly different from those of procedural controls. Additional stressor(s) appeared important, because bacterial communities of Dry and Watered transplants were only marginally different from each other (p = 0.059). The relative abundance of Rhodobacteraceae associated with holdfasts generally correlated with environmental stress with highest abundance associated with F. spiralis and the two high-zone transplant treatments. These findings suggest that the abiotic stressors that shape distributional patterns of host species also affect their bacterial communities.

Reliability of stool microbiome methods for DNA yields and sequencing among infants and young children.

With the emergence of large-scale epidemiologic human microbiome studies, there is a need to understand the reproducibility of microbial DNA sequencing and the impact of specimen collection and processing methods on measures of microbial community composition and structure, with reproducibility studies in infants and young children particularly lacking. Here, we examined batch-to-batch variability and reliability of collection, handling, and processing protocols, testing replicate stool samples from infants and young children using Illumina MiSeq sequencing of the bacterial 16S rRNA gene V4-V5 hypervariable region, evaluating 33 conditions with different protocols and extraction methods. We detected no evidence of batch effects in replicate DNA samples or extractions from the same stool sample. Variability in DNA yield and alpha diversity was observed between the different collection, handling, and processing protocols. However, across all protocols, subject variability was the dominant contributor to microbiome structure, with comparatively little impact of the protocol used. While collection method and DNA extraction kit may affect DNA yield, and correspondingly alpha diversity, our findings suggest that characterization of the structure and composition of the fecal microbiome of infants and young children are reliably measurable by standardized collection, handling, and processing protocols and DNA extraction methods within an individual longitudinal study.