Universal gut microbial relationships in the gut microbiome of wild baboons.

Ecological relationships between bacteria mediate the services that gut microbiomes provide to their hosts. Knowing the overall direction and strength of these relationships is essential to learn how ecology scales up to affect microbiome assembly, dynamics, and host health. However, whether bacterial relationships are generalizable across hosts or personalized to individual hosts is debated. Here, we apply a robust, multinomial logistic-normal modeling framework to extensive time series data (5534 samples from 56 baboon hosts over 13 years) to infer thousands of correlations in bacterial abundance in individual baboons and test the degree to which bacterial abundance correlations are 'universal'. We also compare these patterns to two human data sets. We find that, most bacterial correlations are weak, negative, and universal across hosts, such that shared correlation patterns dominate over host-specific correlations by almost twofold. Further, taxon pairs that had inconsistent correlation signs (either positive or negative) in different hosts always had weak correlations within hosts. From the host perspective, host pairs with the most similar bacterial correlation patterns also had similar microbiome taxonomic compositions and tended to be genetic relatives. Compared to humans, universality in baboons was similar to that in human infants, and stronger than one data set from human adults. Bacterial families that showed universal correlations in human infants were often universal in baboons. Together, our work contributes new tools for analyzing the universality of bacterial associations across hosts, with implications for microbiome personalization, community assembly, and stability, and for designing microbiome interventions to improve host health.

Communities of bacteria living in the guts of humans and other animals perform essential services for their hosts such as digesting food, degrading toxins, or fighting viruses and other bacteria that cause disease. These services emerge from so-called 'ecological' relationships between different species of bacteria. One species, for example, may break down a molecule in human food into another compound that is, in turn, digested by another species into a small molecule that the human gut can absorb and use. The bacteria involved in such a process may become more or less common together in their host. In other situations, some bacteria may have opposing roles to each other, meaning that if one species becomes more abundant it may reduce the level of the other. However, it is not known if relationships between different bacteria are consistent across hosts (i.e., universal) or unique to each host (personalized). In other words, if a pair of bacteria increase and decrease in abundance together in one host, do they do the same in other hosts? Microbes often swap genes with each other to gain new traits; as each host harbors a distinctive set of gut microbes, it may be possible for microbial relationships to change depending on the bacterial species present in a specific environment. To investigate, Roche et al. studied the bacteria in thousands of samples of feces collected from 56 baboons over a 13-year period. These samples came from a long-term research project in Amboseli, Kenya which has been studying a population of wild baboons continuously since 1971. Roche et al. measured the abundance of hundreds of gut bacteria in the feces to understand the relationships between pairs. This revealed that connections between species were largely universal rather than personalized to each baboon. Furthermore, the pairs of bacteria with the strongest positive or negative associations had the most consistent relationships across the baboons. Microbial relationships that have strong effects on the microbiome's composition might therefore be especially universal. Further analyses measuring gut bacteria in human babies also found that relationships between pairs of bacteria were largely universal. Hence, individual species of bacteria may fill similar ecological roles in each host across humans and other primates, and perhaps also in other mammals. These findings suggest that it may be possible to leverage the ecological relationships between bacteria to develop universal therapies for human diseases associated with gut bacteria, such as inflammatory bowel disease or Clostridium difficile infection.

Synchrony and idiosyncrasy in the gut microbiome of wild baboons.

Human gut microbial dynamics are highly individualized, making it challenging to link microbiota to health and to design universal microbiome therapies. This individuality is typically attributed to variation in host genetics, diets, environments and medications but it could also emerge from fundamental ecological forces that shape microbiota more generally. Here, we leverage extensive gut microbial time series from wild baboons-hosts who experience little interindividual dietary and environmental heterogeneity-to test whether gut microbial dynamics are synchronized across hosts or largely idiosyncratic. Despite their shared lifestyles, baboon microbiota were only weakly synchronized. The strongest synchrony occurred among baboons living in the same social group, probably because group members range over the same habitat and simultaneously encounter the same sources of food and water. However, this synchrony was modest compared to each host's personalized dynamics. In support, host-specific factors, especially host identity, explained, on average, more than three times the deviance in longitudinal dynamics compared to factors shared with social group members and ten times the deviance of factors shared across the host population. These results contribute to mounting evidence that highly idiosyncratic gut microbiomes are not an artefact of modern human environments and that synchronizing forces in the gut microbiome (for example, shared environments, diets and microbial dispersal) are not strong enough to overwhelm key drivers of microbiome personalization, such as host genetics, priority effects, horizontal gene transfer and functional redundancy.

Gut microbiome heritability is nearly universal but environmentally contingent.

Relatives have more similar gut microbiomes than nonrelatives, but the degree to which this similarity results from shared genotypes versus shared environments has been controversial. Here, we leveraged 16,234 gut microbiome profiles, collected over 14 years from 585 wild baboons, to reveal that host genetic effects on the gut microbiome are nearly universal. Controlling for diet, age, and socioecological variation, 97% of microbiome phenotypes were significantly heritable, including several reported as heritable in humans. Heritability was typically low (mean = 0.068) but was systematically greater in the dry season, with low diet diversity, and in older hosts. We show that longitudinal profiles and large sample sizes are crucial to quantifying microbiome heritability, and indicate scope for selection on microbiome characteristics as a host phenotype.

Fungal Diversity in Multiple Post-harvest Aged Red Pine Stumps and Their Potential Influence on Heterobasidion Root Rot in Managed Stands Across Minnesota.

Thinning operations that occur in managed red pine (Pinus resinosa) stands, create tree stumps that can serve as a habitat for fungi, especially Heterobasidion irregulare, the cause of a serious root disease. Different fungi can colonize stumps early and the community of fungi can change over time as initial fungal species become replaced. Samples were collected from both the native and non-native range of red pine from stumps that were cut at different time periods. Stumps that were harvested at 0-1, 2-3, 5-6, and 10-12 years before sampling were used to provide data on the diversity of fungi that colonize tree stumps and how these communities can change over time as well as how they influence colonization of H. irregulare. Traditional culturing methods and Illumina MiSeq sequencing were used to identify the fungi in the samples. Of particular interest was Phlebiopsis gigantea, which can colonize cut stumps and prevent H. irregulare from becoming established. Overall, P. gigantea was the most abundant fungus isolated and sequenced via Illumina MiSeq. Results show that Phlebiopsis gigantea was isolated from 90% of all stumps sampled for sites harvested within 3 years of sampling in the native range of red pine compared to 33% in the non-native range. For Illumina MiSeq, 5,940 total amplicon sequence variants (ASVs) were detected. P. gigantea represented 14% of the total reads and composed 19% of the reads in the native range and 8% in non-native range of red pine. Furthermore, P. gigantea represented 38% of the reads for stumps that were harvested within 3 years of sampling in the native range of red pine compared to 14% in the non-native range. These results help demonstrate that a higher amount of P. gigantea is present in the native range of red pine and could be acting as a native biological control agent. Additional fungi, including Resinicium bicolor, Hypochnicium cremicolor, Leptographium spp., and others identified at different cutting times are also discussed. Finally, different diversity indices revealed similar, but slightly higher diversity for southern sites via Shannon and Simpson Diversity indices. Beta diversity demonstrated a similar species composition in stumps harvested at different times with these stumps being grouped together based on harvesting years.

Gut Microbiota Has a Widespread and Modifiable Effect on Host Gene Regulation.

Variation in gut microbiome is associated with wellness and disease in humans, and yet the molecular mechanisms by which this variation affects the host are not well understood. A likely mechanism is that of changing gene regulation in interfacing host epithelial cells. Here, we treated colonic epithelial cells with live microbiota from five healthy individuals and quantified induced changes in transcriptional regulation and chromatin accessibility in host cells. We identified over 5,000 host genes that change expression, including 588 distinct associations between specific taxa and host genes. The taxa with the strongest influence on gene expression alter the response of genes associated with complex traits. Using ATAC-seq, we showed that a subset of these changes in gene expression are associated with changes in host chromatin accessibility and transcription factor binding induced by exposure to gut microbiota. We then created a manipulated microbial community with titrated doses of Collinsella, demonstrating that manipulation of the composition of the microbiome under both natural and controlled conditions leads to distinct and predictable gene expression profiles in host cells. Taken together, our results suggest that specific microbes play an important role in regulating expression of individual host genes involved in human complex traits. The ability to fine-tune the expression of host genes by manipulating the microbiome suggests future therapeutic routes.IMPORTANCE The composition of the gut microbiome has been associated with various aspects of human health, but the mechanism of this interaction is still unclear. We utilized a cellular system to characterize the effect of the microbiome on human gene expression. We showed that some of these changes in expression may be mediated by changes in chromatin accessibility. Furthermore, we validate the role of a specific microbe and show that changes in its abundance can modify the host gene expression response. These results show an important role of gut microbiota in regulating host gene expression and suggest that manipulation of microbiome composition could be useful in future therapies.

Chronic obstructive pulmonary disease upper airway microbiome is associated with select clinical characteristics.

BACKGROUND: Chronic obstructive pulmonary disease (COPD) is an inflammatory lung disorder associated with lung microbiome dysbiosis. Although the upper airway microbiome is the source of the lung microbiome, the relationships between the oral, nasal, and sputum microbiota are incompletely understood. Our objective was to determine features that differentiate the oral, nasal, and sputum microbiome among subjects with stable COPD. METHODS: We recruited 15 current or former smokers to provide oral and sputum samples on day 1. On day 2, another oral sample and a nasal sample were obtained. Each sample and control underwent DNA extraction, 16S V4 rRNA amplification, 16S V4 sequencing, and qPCR of 16S rRNA. Data were analyzed using dada2 and R. RESULTS: Most (14 of 15) subjects were male with a mean age of 65.2. One subject had no pulmonary obstruction, while 5 had mild COPD, 7 had moderate COPD, and 2 had severe COPD. Three subjects (20%) were current tobacco users and 2 subjects (13%) used inhaled corticosteroids (ICS). Subjects had a mean of 49.1 pack-years of tobacco exposure. Bacterial biomass was associated with anatomic site, but no differences in biomass were observed with age, FEV1 percent predicted (FEV1pp), ICS use, smoking status, or edentulous state. Shannon index was associated with site (lower nasal diversity than oral and sputum diversity, p<0.001), but not age, ICS use, FEV1pp, tobacco use, or edentulous state. ß-diversity was illustrated by principal coordinate analysis using Bray-Curtis dissimilarity and PERMANOVA analyses, showing sample clustering by anatomic site (p = 0.001) with nasal samples forming a cluster separate from the combined oral wash samples and sputum samples. Clustering was also observed with ICS use (p = 0.029) and edentulous state (p = 0.019), while FEV1pp and current tobacco use were not significant. In an amplicon sequencing variant (ASV)-level analysis of oral samples using a linear regression model with Benjamini-Hochberg correction at an FDR<0.10, 10 ASVs were associated with age while no ASVs were associated with FEV1pp or smoking status. Sputum sample analysis demonstrated that 51 ASVs (25 unique genera) were associated with age, 61 ASVs (32 genera) were associated with FEV1pp, and no ASVs were associated with smoking status. In a combined dataset, the frequent exacerbator phenotype, rather than ICS use, was associated with decreased sputum Shannon diversity. CONCLUSIONS: Among the upper airway microbiota of COPD subjects, anatomic site was associated with bacterial biomass, Shannon diversity, and ß-diversity. ICS use and edentulous state were both associated with ß-diversity. Age was associated with taxa relative abundance in oral and sputum samples, while FEV1pp was associated with taxa relative abundance in sputum samples only.

Chronic obstructive pulmonary disease upper airway microbiota alpha diversity is associated with exacerbation phenotype: a case-control observational study.

BACKGROUND: Chronic obstructive pulmonary disease (COPD) frequent exacerbators (FE) suffer increased morbidity and mortality compared to infrequent exacerbators (IE). The association between the oral and sputum microbiota and exacerbation phenotype is not well defined. The objective of this study was to determine key features that differentiate the oral and sputum microbiota of FEs from the microbiota of IEs during periods of clinical stability. METHODS: We recruited 11 FE and 11 IE who had not used antibiotics or systemic corticosteroids in the last 1 month. Subjects provided oral wash and sputum samples, which underwent 16S V4 MiSeq sequencing and qPCR of 16S rRNA. Data were analyzed using Dada2 and R. RESULTS: FE and IE were similar in terms of age, FEV1 percent predicted (FEV1pp), pack-years of tobacco exposure, and St. George's Respiratory Questionnaire score. 16S copy numbers were significantly greater in sputum vs. oral wash (p = 0.01), but phenotype was not associated with copy number. Shannon diversity was significantly greater in oral samples compared to sputum (p = 0.001), and IE samples were more diverse than FE samples (p < 0.001). Sputum samples from FE had more Haemophilus and Moraxella compared to IE sputum samples, due to dominance of these COPD-associated taxa in three FE sputum samples. Amplicon sequencing variant (ASV)-level analysis of sputum samples revealed one ASV (Actinomyces) was significantly more abundant in IE vs. FE sputum (padj = 0.048, Wilcoxon rank-sum test), and this persisted after controlling for FEV1pp. Principal coordinate analysis using Bray-Curtis distance with PERMANOVA analyses demonstrated clustering by anatomic site, phenotype, inhaled corticosteroid use, current tobacco use, COPD severity, and last professional dental cleaning. CONCLUSIONS: FE have less diverse oral and sputum microbiota than IE. Actinomyces was significantly more abundant in IE sputum than FE sputum. The oral and sputum microbiota of COPD subjects cluster based on multiple clinical factors, including exacerbation phenotype. Even during periods of clinical stability, the frequent exacerbator phenotype is associated with decreased alpha diversity, beta-diversity clustering, and changes in taxonomic abundance.

The lung tissue microbiota of mild and moderate chronic obstructive pulmonary disease.

BACKGROUND: Oral taxa are often found in the chronic obstructive pulmonary disease (COPD) lung microbiota, but it is not clear if this is due to a physiologic process such as aspiration or experimental contamination at the time of specimen collection. METHODS: Microbiota samples were obtained from nine subjects with mild or moderate COPD by swabbing lung tissue and upper airway sites during lung lobectomy. Lung specimens were not contaminated with upper airway taxa since they were obtained surgically. The microbiota were analyzed with 16S rRNA gene qPCR and 16S rRNA gene hypervariable region 3 (V3) sequencing. Data analyses were performed using QIIME, SourceTracker, and R. RESULTS: Streptococcus was the most common genus in the oral, bronchial, and lung tissue samples, and multiple other taxa were present in both the upper and lower airways. Each subject's own bronchial and lung tissue microbiota were more similar to each other than were the bronchial and lung tissue microbiota of two different subjects (permutation test, p = 0.0139), indicating more within-subject similarity than between-subject similarity at these two lung sites. Principal coordinate analysis of all subject samples revealed clustering by anatomic sampling site (PERMANOVA, p = 0.001), but not by subject. SourceTracker analysis found that the sources of the lung tissue microbiota were 21.1% (mean) oral microbiota, 8.7% nasal microbiota, and 70.1% unknown. An analysis using the neutral theory of community ecology revealed that the lung tissue microbiota closely reflects the bronchial, oral, and nasal microbiota (immigration parameter estimates 0.69, 0.62, and 0.74, respectively), with some evidence of ecologic drift occurring in the lung tissue. CONCLUSION: This is the first study to evaluate the mild-moderate COPD lung tissue microbiota without potential for upper airway contamination of the lung samples. In our small study of subjects with COPD, we found oral and nasal bacteria in the lung tissue microbiota, confirming that aspiration is a source of the COPD lung microbiota.

Prebiotic Dietary Fiber and Gut Health: Comparing the in Vitro Fermentations of Beta-Glucan, Inulin and Xylooligosaccharide.

Prebiotic dietary fiber supplements are commonly consumed to help meet fiber recommendations and improve gastrointestinal health by stimulating beneficial bacteria and the production of short-chain fatty acids (SCFAs), molecules beneficial to host health. The objective of this research project was to compare potential prebiotic effects and fermentability of five commonly consumed fibers using an in vitro fermentation system measuring changes in fecal microbiota, total gas production and formation of common SCFAs. Fecal donations were collected from three healthy volunteers. Materials analyzed included: pure beta-glucan, Oatwell (commercially available oat-bran containing 22% oat ß-glucan), xylooligosaccharides (XOS), WholeFiber (dried chicory root containing inulin, pectin, and hemi/celluloses), and pure inulin. Oatwell had the highest production of propionate at 12 h (4.76 µmol/mL) compared to inulin, WholeFiber and XOS samples (p < 0.03). Oatwell's effect was similar to those of the pure beta-glucan samples, both samples promoted the highest mean propionate production at 24 h. XOS resulted in a significant increase in the genus Bifidobacterium after 24 h of fermentation (0 h:0.67 OTUs (operational taxonomic unit); 24 h:5.22 OTUs; p = 0.038). Inulin and WholeFiber increased the beneficial genus Collinsella, consistent with findings in clinical studies. All analyzed compounds were fermentable and promoted the formation of beneficial SCFAs.

Structure of bacterial communities in soil following cover crop and organic fertilizer incorporation.

Incorporation of organic material into soils is an important element of organic farming practices that can affect the composition of the soil bacterial communities that carry out nutrient cycling and other functions crucial to crop health and growth. We conducted a field experiment to determine the effects of cover crops and fertilizers on bacterial community structure in agricultural soils under long-term organic management. Illumina sequencing of 16S rDNA revealed diverse communities comprising 45 bacterial phyla in corn rhizosphere and bulk field soil. Community structure was most affected by location and by the rhizosphere effect, followed by sampling time and amendment treatment. These effects were associated with soil physicochemical properties, including pH, moisture, organic matter, and nutrient levels. Treatment differences were apparent in bulk and rhizosphere soils at the time of peak corn growth in the season following cover crop and fertilizer application. Cover crop and fertilizer treatments tended to lower alpha diversity in early season samples. However, winter rye, oilseed radish, and buckwheat cover crop treatments increased alpha diversity in some later season samples compared to a no-amendment control. Fertilizer treatments and some cover crops decreased relative abundance of members of the ammonia-oxidizing family Nitrosomonadaceae. Pelleted poultry manure and Sustane® (a commercial fertilizer) decreased the relative abundance of Rhizobiales. Our data point to a need for future research exploring how (1) cover crops influence bacterial community structure and functions, (2) these effects differ with biomass composition and quantity, and (3) existing soil conditions and microbial community composition influence how soil microbial populations respond to agricultural management practices.

Systematic improvement of amplicon marker gene methods for increased accuracy in microbiome studies.

Amplicon-based marker gene surveys form the basis of most microbiome and other microbial community studies. Such PCR-based methods have multiple steps, each of which is susceptible to error and bias. Variance in results has also arisen through the use of multiple methods of next-generation sequencing (NGS) amplicon library preparation. Here we formally characterized errors and biases by comparing different methods of amplicon-based NGS library preparation. Using mock community standards, we analyzed the amplification process to reveal insights into sources of experimental error and bias in amplicon-based microbial community and microbiome experiments. We present a method that improves on the current best practices and enables the detection of taxonomic groups that often go undetected with existing methods.

Associations between soil bacterial community structure and nutrient cycling functions in long-term organic farm soils following cover crop and organic fertilizer amendment.

Agricultural management practices can produce changes in soil microbial populations whose functions are crucial to crop production and may be detectable using high-throughput sequencing of bacterial 16S rRNA. To apply sequencing-derived bacterial community structure data to on-farm decision-making will require a better understanding of the complex associations between soil microbial community structure and soil function. Here 16S rRNA sequencing was used to profile soil bacterial communities following application of cover crops and organic fertilizer treatments in certified organic field cropping systems. Amendment treatments were hairy vetch (Vicia villosa), winter rye (Secale cereale), oilseed radish (Raphanus sativus), buckwheat (Fagopyrum esculentum), beef manure, pelleted poultry manure, Sustane(®) 8-2-4, and a no-amendment control. Enzyme activities, net N mineralization, soil respiration, and soil physicochemical properties including nutrient levels, organic matter (OM) and pH were measured. Relationships between these functional and physicochemical parameters and soil bacterial community structure were assessed using multivariate methods including redundancy analysis, discriminant analysis, and Bayesian inference. Several cover crops and fertilizers affected soil functions including N-acetyl-ß-d-glucosaminidase and ß-glucosidase activity. Effects, however, were not consistent across locations and sampling timepoints. Correlations were observed among functional parameters and relative abundances of individual bacterial families and phyla. Bayesian analysis inferred no directional relationships between functional activities, bacterial families, and physicochemical parameters. Soil functional profiles were more strongly predicted by location than by treatment, and differences were largely explained by soil physicochemical parameters. Composition of soil bacterial communities was predictive of soil functional profiles. Differences in soil function were better explained using both soil physicochemical test values and bacterial community structure data than using soil tests alone. Pursuing a better understanding of bacterial community composition and how it is affected by farming practices is a promising avenue for increasing our ability to predict the impact of management practices on important soil functions.

Sediments and Soils Act as Reservoirs for Taxonomic and Functional Bacterial Diversity in the Upper Mississippi River.

In this study, we utilized Illumina next-generation sequencing of 16S rDNA to characterize the bacterial communities in water, sediments, and soils at four sites along the Mississippi River and Minnesota River, in Minnesota, in order to evaluate community exchanges between these habitats. Communities in water and sediment were hypothesized to show greater taxonomic similarity than those in soil, while microbial communities in sediment and soil would show greater functional similarity. Habitat-specific communities showed significant differences in phylogenetic structure and ß-diversity (P < 0.001), but site-specific differences in community structures within a single habitat type did not differ greatly (P ≥ 0.083). Community exchange among habitats generally influenced < 5% of the total community composition in a single sample, with the exception of the sediment community at the Minnesota River site, which contributed to a mean of 14% of the microbial community in the water column. Communities from all habitat types were significantly correlated with each other (r = 0.44-0.64, P ≤ 0.004). Furthermore, approximately 33% of the taxonomic units were found in all samples and comprised at least 40% of the bacterial community. Functional annotation of shotgun sequencing data revealed similar functional profiles for sediment and soil communities that were distinct from those in the water. Results of this study suggest that sediments, when disturbed, contribute significantly to bacterial communities in the water and that a core bacterial community may be supported in the soils and sediments. Furthermore, a high degree of functional redundancy results in similar functional profiles in sediment and soil communities.

High-throughput functional screening reveals low frequency of antibiotic resistance genes in DNA recovered from the Upper Mississippi River.

In this study, we determined the frequency of antibiotic resistance genes (ARGs) in the Upper Mississippi River using a high-throughput, functional, metagenomic screening procedure. Fosmid libraries containing ∼10,000 clones were screened for resistance to ampicillin, cephalothin, kanamycin, and tetracycline. We hypothesized that nutrient concentrations, land cover type, and taxonomic community composition may select for ARGs. Resistance to ampicillin, cephalothin, and kanamycin was low (<1.00%), and no resistance to tetracycline was detected. Ammonium and total dissolved solids (TDS) concentrations were correlated with kanamycin and cephalothin resistances (r=0.617 and -0.449, P=0.002 and 0.036, respectively). Cephalothin resistance was also positively correlated with the percentage of forested land cover (r=0.444, P=0.039). Only the candidate division OD1, among 35 phyla identified, was correlated with ampicillin resistance (r=0.456, P=0.033), suggesting that minority members of the community may be responsible for dissemination of ARGs in this ecosystem. Results of this study suggest that ammonium and TDS may be involved in a complex selection process for ARGs. Furthermore, we suggest that minority species, potentially contributed in low numbers from sediment and biofilm reservoirs, may be the primary carriers of ARGs in this riverine system.

Evaluation of water sampling methodologies for amplicon-based characterization of bacterial community structure.

Reduction in costs of next-generation sequencing technologies has allowed unprecedented characterization of bacterial communities from environmental samples including aquatic ecosystems. However, the extent to which extrinsic factors including sampling volume, sample replication, DNA extraction kits, and sequencing target affect the community structure inferred are poorly explored. Here, triplicate 1, 2, and 6L volume water samples from the Upper Mississippi River were processed to determine variation among replicates and sample volumes. Replicate variability significantly influenced differences in the community α-diversity (P=0.046), while volume significantly changed ß-diversity (P=0.037). Differences in phylogenetic and taxonomic community structure differed both among triplicate samples and among the volumes filtered. Communities from 2L and 6L water samples showed similar clustering via discriminant analysis. To assess variation due to DNA extraction method, DNA was extracted from triplicate cell pellets from four sites along the Upper Mississippi River using the Epicentre Metagenomic DNA Isolation Kit for Water and MoBio PowerSoil kit. Operational taxonomic units representing ≤14% of sequence reads differed significantly among all sites and extraction kits used, although differences in diversity and community coverage were not significant (P≥0.057). Samples characterized using only the V6 region had significantly higher coverage and lower richness and α-diversity than those characterized using V4-V6 regions (P<0.001). Triplicate sampling of at least 2L of water provides robust representation of community variability, and these results indicate that DNA extraction kit and sequencing target displayed taxonomic biases that did not affect the overall biological conclusions drawn.

Frequencies of heavy metal resistance are associated with land cover type in the Upper Mississippi River.

Taxonomic compositions of freshwater bacterial communities have been well-characterized via metagenomic and amplicon-based approaches, especially next-generation sequencing. However, functional diversity of these communities remains less well-studied. Various anthropogenic sources are known to impact the bacterial community composition in freshwater riverine systems and potentially alter functional diversity. In this study, high-throughput functional screening of large (~10,000 clones) fosmid libraries representing communities in the Upper Mississippi River revealed low frequencies of resistance to heavy metals in the following order: Mn2+>Cr3+>Zn2+>Cd2+>Hg2+. No resistance to Cu2+ was detected. Significant, but weak, correlations were observed between resistance frequencies of Cd and Cr with developed land cover (r2=0.08, P=0.016 and r=0.07, P=0.037, respectively). While discriminant function analyses further supported these associations, redundancy analysis further indicated associations with forested land cover and greater resistance to Hg and Zn. Nutrient and metal ion concentrations and abundances of bacterial orders were poorly correlated with heavy metal resistance, except for an association of Pseudomonadales abundance and resistance to Hg and Zn. Taken together, results of this study suggest that allochthonous bacteria contributed from specific land cover types influence the patterns of metal resistance throughout this river.

Species sorting and seasonal dynamics primarily shape bacterial communities in the Upper Mississippi River.

Bacterial community structure (BCS) in freshwater ecosystems varies seasonally and due to physicochemical gradients, but metacommunity structure of a major river remains understudied. Here we characterize the BCS along the Mississippi River and contributing rivers in Minnesota over three years using Illumina next-generation sequencing, to determine how changes in environmental conditions as well as inputs from surrounding land and confluences impacted community structure. Contributions of sediment to water microbial diversity were also evaluated. Long-term variation in community membership was observed, and significant shifts in relative abundances of major freshwater taxa, including α-Proteobacteria, Burkholderiales, and Actinomycetales, were observed due to temporal and spatial variations. Environmental parameters (e.g. temperature, rainfall, and nutrient concentrations) primarily contributed to differences in phyla abundances (88% of variance), with minimal influence from spatial distance alone (<1% of variance). Furthermore, an annually-recurrent BCS was observed in late summer, further suggesting that seasonal dynamics strongly influence community composition. Sediment communities differed from those in the water, but contributed up to 50% to community composition in the water column. Among water sampling sites, 34% showed significant variability in BCS of replicate samples indicating variability among riverine communities due to heterogeneity in the water column. Results of this study highlight the need for a better understanding of spatial and temporal variations in riverine bacterial diversity associated with physicochemical gradients and reveal how communities in sediments, and potentially other environmental reservoirs, impact waterborne BCS. Techniques used in this study may prove useful to determine sources of microbes from sediments and soils to waterways, which will facilitate best management practices and total maximum daily load determinations.

Bacterial community structure is indicative of chemical inputs in the Upper Mississippi River.

Local and regional associations between bacterial communities and nutrient and chemical concentrations were assessed in the Upper Mississippi River in Minnesota to determine if community structure was associated with discrete types of chemical inputs associated with different land cover. Bacterial communities were characterized by Illumina sequencing of the V6 region of 16S rDNA and compared to >40 chemical and nutrient concentrations. Local bacterial community structure was shaped primarily by associations among bacterial orders. However, order abundances were correlated regionally with nutrient and chemical concentrations, and were also related to major land coverage types. Total organic carbon and total dissolved solids were among the primary abiotic factors associated with local community composition and co-varied with land cover. Escherichia coli concentration was poorly related to community composition or nutrient concentrations. Abundances of 14 bacterial orders were related to land coverage type, and seven showed significant differences in abundance (P ≤ 0.046) between forested or anthropogenically-impacted sites. This study identifies specific bacterial orders that were associated with chemicals and nutrients derived from specific land cover types and may be useful in assessing water quality. Results of this study reveal the need to investigate community dynamics at both the local and regional scales and to identify shifts in taxonomic community structure that may be useful in determining sources of pollution in the Upper Mississippi River.

Core functional traits of bacterial communities in the Upper Mississippi River show limited variation in response to land cover.

Taxonomic characterization of environmental microbial communities via high-throughput DNA sequencing has revealed that patterns in microbial biogeography affect community structure. However, shifts in functional diversity related to variation in taxonomic composition are poorly understood. To overcome limitations due to the prohibitive cost of high-depth metagenomic sequencing, tools to infer functional diversity based on phylogenetic distributions of functional traits have been developed. In this study we characterized functional microbial diversity at 11 sites along the Mississippi River in Minnesota using both metagenomic sequencing and functional-inference-based (PICRUSt) approaches. This allowed us to determine how distance and variation in land cover throughout the river influenced the distribution of functional traits, as well as to validate PICRUSt inferences. The distribution and abundance of functional traits, by metagenomic analysis, were similar among sites, with a median standard deviation of 0.0002% among tier 3 functions in KEGG. Overall inferred functional variation was significantly different (P ≤ 0.035) between two water basins surrounded by agricultural vs. developed land cover, and abundances of bacterial orders that correlated with functional traits by metagenomic analysis were greater where abundances of the trait were inferred to be higher. PICRUSt inferences were significantly correlated (r = 0.147, P = 1.80 × 10(-30)) with metagenomic annotations. Discrepancies between metagenomic and PICRUSt taxonomic-functional relationships, however, suggested potential functional redundancy among abundant and rare taxa that impeded the ability to accurately assess unique functional traits among rare taxa at this sequencing depth. Results of this study suggest that a suite of "core functional traits" is conserved throughout the river and distributions of functional traits, rather than specific taxa, may shift in response to environmental heterogeneity.