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As agriculture industrializes, concentrated animal feeding operations (CAFOs) are becoming more common. Feces from CAFOs is often used as fertilizer on fields. However, little is known about the effects manure has on the soil microbiome, which is an important aspect of soil health and fertility. In addition, due to the subtherapeutic levels of antibiotics necessary to keep the animals healthy, CAFO manure has elevated levels of antibiotic resistant bacteria. Using 16s rRNA high-throughput sequencing and qPCR, this study sought to determine the impact of swine CAFO manure application on both the soil microbiome and abundance of select antibiotic resistance genes (ARGs) and mobile element genes (erm(B), erm(C), sul1, str(B), intI1, IncW repA) in agricultural soil over the fall and spring seasons. We found the manure community to be distinct from the soil community, with a majority of bacteria belonging to Bacteroidetes and Firmicutes. The soil samples had more diverse communities dominated by Acidobacteria, Actinobacteria, Proteobacteria, Verrucomicrobia, and unclassified bacteria. We observed significant differences in the soil microbiome between all time points, except between the spring samples. However, by tracking manure associated taxa, we found the addition of the manure microbiome to be a minor driver of the shift. Of the measured genes, manure application only significantly increased the abundance of erm(B) and erm(C) which remained elevated in the spring. These results suggest bacteria in the manure do not survive well in soil and that ARG dynamics in soil following manure application vary by resistance gene.

The evolutionary and environmental factors that shape fungal biogeography are incompletely understood. Here, we assemble a large dataset consisting of previously generated mycobiome data linked to specific geographical locations across the world. We use this dataset to describe the distribution of fungal taxa and to look for correlations with different environmental factors such as climate, soil and vegetation variables. Our meta-study identifies climate as an important driver of different aspects of fungal biogeography, including the global distribution of common fungi as well as the composition and diversity of fungal communities. In our analysis, fungal diversity is concentrated at high latitudes, in contrast with the opposite pattern previously shown for plants and other organisms. Mycorrhizal fungi appear to have narrower climatic tolerances than pathogenic fungi. We speculate that climate change could affect ecosystem functioning because of the narrow climatic tolerances of key fungal taxa.

Butyrate-producing bacteria have recently gained attention, since they are important for a healthy colon and when altered contribute to emerging diseases, such as ulcerative colitis and type II diabetes. This guild is polyphyletic and cannot be accurately detected by 16S rRNA gene sequencing. Consequently, approaches targeting the terminal genes of the main butyrate-producing pathway have been developed. However, since additional pathways exist and alternative, newly recognized enzymes catalyzing the terminal reaction have been described, previous investigations are often incomplete. We undertook a broad analysis of butyrate-producing pathways and individual genes by screening 3,184 sequenced bacterial genomes from the Integrated Microbial Genome database. Genomes of 225 bacteria with a potential to produce butyrate were identified, including many previously unknown candidates. The majority of candidates belong to distinct families within the Firmicutes , but members of nine other phyla, especially from Actinobacteria , Bacteroidetes , Fusobacteria , Proteobacteria , Spirochaetes , and Thermotogae , were also identified as potential butyrate producers. The established gene catalogue (3,055 entries) was used to screen for butyrate synthesis pathways in 15 metagenomes derived from stool samples of healthy individuals provided by the HMP (Human Microbiome Project) consortium. A high percentage of total genomes exhibited a butyrate-producing pathway (mean, 19.1%; range, 3.2% to 39.4%), where the acetyl-coenzyme A (CoA) pathway was the most prevalent (mean, 79.7% of all pathways), followed by the lysine pathway (mean, 11.2%). Diversity analysis for the acetyl-CoA pathway showed that the same few firmicute groups associated with several Lachnospiraceae and Ruminococcaceae were dominating in most individuals, whereas the other pathways were associated primarily with Bacteroidetes. IMPORTANCE Microbiome research has revealed new, important roles of our gut microbiota for maintaining health, but an understanding of effects of specific microbial functions on the host is in its infancy, partly because in-depth functional microbial analyses are rare and publicly available databases are often incomplete/misannotated. In this study, we focused on production of butyrate, the main energy source for colonocytes, which plays a critical role in health and disease. We have provided a complete database of genes from major known butyrate-producing pathways, using in-depth genomic analysis of publicly available genomes, filling an important gap to accurately assess the butyrate-producing potential of complex microbial communities from “-omics”-derived data. Furthermore, a reference data set containing the abundance and diversity of butyrate synthesis pathways from the healthy gut microbiota was established through a metagenomics-based assessment. This study will help in understanding the role of butyrate producers in health and disease and may assist the development of treatments for functional dysbiosis. Microbiome research has revealed new, important roles of our gut microbiota for maintaining health, but an understanding of effects of specific microbial functions on the host is in its infancy, partly because in-depth functional microbial analyses are rare and publicly available databases are often incomplete/misannotated. In this study, we focused on production of butyrate, the main energy source for colonocytes, which plays a critical role in health and disease. We have provided a complete database of genes from major known butyrate-producing pathways, using in-depth genomic analysis of publicly available genomes, filling an important gap to accurately assess the butyrate-producing potential of complex microbial communities from “-omics”-derived data. Furthermore, a reference data set containing the abundance and diversity of butyrate synthesis pathways from the healthy gut microbiota was established through a metagenomics-based assessment. This study will help in understanding the role of butyrate producers in health and disease and may assist the development of treatments for functional dysbiosis.

Lignin is an abundant and complex plant polymer that may limit litter decomposition, yet lignin is sometimes a minor constituent of soil organic carbon (SOC). Accounting for diversity in soil characteristics might reconcile this apparent contradiction. Tracking decomposition of a lignin/litter mixture and SOC across different North American mineral soils using lab and field incubations, here we show that cumulative lignin decomposition varies 18-fold among soils and is strongly correlated with bulk litter decomposition, but not SOC decomposition. Climate legacy predicts decomposition in the lab, and impacts of nitrogen availability are minor compared with geochemical and microbial properties. Lignin decomposition increases with some metals and fungal taxa, whereas SOC decomposition decreases with metals and is weakly related with fungi. Decoupling of lignin and SOC decomposition and their contrasting biogeochemical drivers indicate that lignin is not necessarily a bottleneck for SOC decomposition and can explain variable contributions of lignin to SOC among ecosystems. Lignin’s contribution to soil organic carbon (SOC) is contentious. The authors find a decoupling of lignin and SOC decomposition and their contrasting relationships with geochemical and microbial factors, addressing a long-standing controversy.

Antibiotics are administered to livestock in animal feeding operations (AFOs) for the control, prevention, and treatment of disease. Manure from antibiotic treated livestock contains unmetabolized antibiotics that provide selective pressure on bacteria, facilitating the expression of anti-microbial resistance (AMR). Manure application on row crops is an agronomic practice used by growers to meet crop nutrient needs; however, it can be a source of AMR to the soil and water environment. This study in central Iowa aims to directly compare AMR indicators in outlet runoff from two adjacent (221 to 229 ha) manured and non-manured catchments (manure comparison), and among three catchments (600 to 804 ha) with manure influence, no known manure application (control), and urban influences (mixed land use comparison). Monitored AMR indicators included antibiotic resistance genes (ARGs) ermB, ermF (macrolide), tetA, tetM, tetO, tetW (tetracycline), sul1, sul2 (sulfonamide), aadA2 (aminoglycoside), vgaA, and vgaB (pleuromutilin), and tylosin and tetracycline resistant enterococci bacteria. Results of the manure comparison showed significantly higher (p<0.05) tetracycline and tylosin resistant bacteria from the catchment with manure application in 2017, but no differences in 2018, possibly due to changes in antibiotic use resulting from the Veterinary Feed Directive. Moreover, the ARG analysis indicated a larger diversity of ARGs at the manure amended catchment. The mixed land use comparison showed the manure amended catchment had significantly higher (p<0.05) tetracycline resistant bacteria in 2017 and significantly higher tylosin resistant bacteria in 2017 and 2018 than the urban influenced catchment. The urban influenced catchment had significantly higher ermB concentrations in both sampling years, however the manure applied catchment runoff consisted of higher relative abundance of total ARGs. Additionally, both catchments showed higher AMR indicators compared to the control catchment. This study identifies four ARGs that might be specific to AMR as a result of agricultural sources (tetM, tetW, sul1, sul2) and optimal for use in watershed scale monitoring studies for tracking resistance in the environment.

Foam accumulation in swine manure deep-pits has been linked to explosions and flash fires that pose devastating threats to humans and livestock. It is clear that methane accumulation within these pits is the fuel for the fire; it is not understood what microbial drivers cause the accumulation and stabilization of methane. Here, we conducted a 13-month field study to survey the physical, chemical, and biological changes of pit-manure across 46 farms in Iowa. Our results showed that an increased methane production rate was associated with less digestible feed ingredients, suggesting that diet influences the storage pit’s microbiome. Targeted sequencing of the bacterial 16S rRNA and archaeal mcrA genes was used to identify microbial communities’ role and influence. We found that microbial communities in foaming and non-foaming manure were significantly different, and that the bacterial communities of foaming manure were more stable than those of non-foaming manure. Foaming manure methanogen communities were enriched with uncharacterized methanogens whose presence strongly correlated with high methane production rates. We also observed strong correlations between feed ration, manure characteristics, and the relative abundance of specific taxa, suggesting that manure foaming is linked to microbial community assemblage driven by efficient free long-chain fatty acid degradation by hydrogenotrophic methanogenesis.

The high-throughput antibiotic resistance gene (ARG) qPCR array, initially published in 2012, is increasingly used to quantify resistance and mobile determinants in environmental matrices. Continued utility of the array; however, necessitates improvements such as removing or redesigning questionable primer sets, updating targeted genes and coverage of available sequences. Towards this goal, a new primer design tool (EcoFunPrimer) was used to aid in identification of conserved regions of diverse genes. The total number of assays used for diverse genes was reduced from 91 old primer sets to 52 new primer sets, with only a 10% loss in sequence coverage. While the old and new array both contain 384 primer sets, a reduction in old primer sets permitted 147 additional ARGs and mobile genetic elements to be targeted. Results of validating the updated array with a mock community of strains resulted in over 98% of tested instances incurring true positive/negative calls. Common queries related to sensitivity, quantification and conventional data analysis (e.g. Ct cutoff value, and estimated genomic copies without standard curves) were also explored. A combined list of new and previously used primer sets is provided with a recommended set based on redesign of primer sets and results of validation.

Bacteria in the honeybee gut are a well-recognized factor affecting bee health. However, the primary focus of this research has been the hindgut, while the crop, or honey stomach, is assumed to be dominated by environmentally acquired transient taxa that matter little to the bees. To evaluate this assumption, we examined bacterial taxa in the crop and mouth of Apis mellifera and A. cerana japonica foragers and in the nectar of Prunus mume flowers visited by the bees in the Minabe-Tanabe region of Japan. We found that in bacterial composition, the crop was distinct from both the mouth and the nectar, whereas mouth and nectar samples were indistinguishable. Furthermore, the crop remained similar in bacterial composition and diversity, while the mouth showed a sharp drop in alpha diversity and a large increase in beta diversity, from summer to winter. These results refute the conventional assumption, suggesting instead that the crop contains a conserved bacterial community largely distinct from environmental taxa. We also found that strains of a crop-associated species, Apilactobacillus kunkeei , could be season- and host species-specific. Together, these findings suggest that crop-associated bacterial communities should be studied further to better understand the relationship between honeybees and their gut bacteria.

Deep sequencing has enabled the investigation of a wide range of environmental microbial ecosystems, but the high memory requirements for de novo assembly of short-read shotgun sequencing data from these complex populations are an increasingly large practical barrier. Here we introduce a memory-efficient graph representation with which we can analyze the k -mer connectivity of metagenomic samples. The graph representation is based on a probabilistic data structure, a Bloom filter, that allows us to efficiently store assembly graphs in as little as 4 bits per k -mer, albeit inexactly. We show that this data structure accurately represents DNA assembly graphs in low memory. We apply this data structure to the problem of partitioning assembly graphs into components as a prelude to assembly, and show that this reduces the overall memory requirements for de novo assembly of metagenomes. On one soil metagenome assembly, this approach achieves a nearly 40-fold decrease in the maximum memory requirements for assembly. This probabilistic graph representation is a significant theoretical advance in storing assembly graphs and also yields immediate leverage on metagenomic assembly.

A database of curated genomes is needed to better assess soil microbial communities and their processes associated with differing land management and environmental impacts. Interpreting soil metagenomic datasets with existing sequence databases is challenging because these datasets are biased towards medical and biotechnology research and can result in misleading annotations. We have curated a database of 928 genomes of soil-associated organisms (888 bacteria, 34 archaea, and 6 fungi). Using this database as a representation of the current state of knowledge of soil microbes that are well-characterized, we evaluated its composition and compared it to broader microbial databases, specifically NCBI’s RefSeq, as well as 3,035 publicly available soil amplicon datasets. These comparisons identified phyla and functions that are enriched in soils as well as those that may be underrepresented in RefSoil. For example, RefSoil was observed to have increased representation of Firmicutes despite its low abundance in soil environments and also lacked representation of Acidobacteria and Verrucomicrobia, which are abundant in soils. Our comparison of RefSoil to soil amplicon datasets allowed us to identify targets that if cultured or sequenced would significantly increase the biodiversity represented within RefSoil. To demonstrate the opportunities to access these underrepresented targets, we employed single cell genomics in a pilot experiment to recover 14 genomes from the \"most wanted\" list, which improved RefSoil's representation of EMP sequences by 7% by abundance. This effort demonstrates the value of RefSoil in the guidance of future research efforts and the capability of single cell genomics as a practical means to fill the existing genomic data gaps.