Summary Ancient and diverse antibiotic resistance genes (ARGs) have previously been identified from soil1–3, including genes identical to those in human pathogens4. Despite the apparent overlap between soil and clinical resistomes4–6, factors influencing ARG composition in soil and their movement between genomes and habitats remain largely unknown3. General metagenome functions often correlate with the underlying structure of bacterial communities7–12. However, ARGs are hypothesized to be highly mobile4,5,13, prompting speculation that resistomes may not correlate with phylogenetic signatures or ecological divisions13,14. To investigate these relationships, we performed functional metagenomic selections for resistance to 18 antibiotics from 18 agricultural and grassland soils. The 2895 ARGs we discovered were predominantly novel, and represent all major resistance mechanisms15. We demonstrate that distinct soil types harbor distinct resistomes, and that nitrogen fertilizer amendments strongly influenced soil ARG content. Resistome composition also correlated with microbial phylogenetic and taxonomic structure, both across and within soil types. Consistent with this strong correlation, mobility elements syntenic with ARGs were rare in soil compared to sequenced pathogens, suggesting that ARGs in the soil may not transfer between bacteria as readily as is observed in the clinic. Together, our results indicate that bacterial community composition is the primary determinant of soil ARG content, challenging previous hypotheses that horizontal gene transfer effectively decouples resistomes from phylogeny13,14.

Summary Antibiotic-resistant infections annually claim hundreds of thousands of lives worldwide. This problem is exacerbated by resistance gene exchange between pathogens and benign microbes from diverse habitats. Mapping resistance gene dissemination between humans and their environment is a public health priority. We characterized the bacterial community structure and resistance exchange networks of hundreds of interconnected human fecal and environmental samples from two low-income Latin American communities. We found that resistomes across habitats are generally structured by bacterial phylogeny along ecological gradients, but identified key resistance genes that cross habitat boundaries and determined their association with mobile genetic elements. We also assessed the effectiveness of widely-used excreta management strategies in reducing fecal bacteria and resistance genes in these settings representative of low- and middle-income countries. Our results lay the foundation for quantitative risk assessment and surveillance of resistance dissemination across interconnected habitats in settings representing over two-thirds of the world’s population.

Summary Enzymes capable of inactivating tetracycline are paradoxically rare compared with enzymes that inactivate other natural product antibiotics. We describe a family of flavoenzymes, previously unrecognizable as resistance genes, which are capable of degrading tetracycline antibiotics. From soil functional metagenomic selections, we discovered nine genes that confer high-level tetracycline resistance by enzymatic inactivation. We also demonstrate that a tenth enzyme, an uncharacterized homolog in the human pathogen Legionella longbeachae, similarly inactivates tetracycline. These enzymes catalyze the oxidation of tetracyclines in vitro both by known mechanisms and via previously undescribed activity. Tetracycline inactivation genes were identified in diverse soil types, encompass substantial sequence diversity, and are adjacent to genes implicated in horizontal gene transfer. Because tetracycline inactivation is scarcely observed in hospitals, these enzymes may fill an empty niche in pathogenic organisms, and should therefore be monitored for their dissemination potential into the clinic.

Emerging antibiotic resistance threatens human health. Gut microbes are an epidemiologically important reservoir of resistance genes (resistome), yet prior studies indicate that the true diversity of gut-associated resistomes has been underestimated. To deeply characterize the pediatric gut-associated resistome, we created metagenomic recombinant libraries in an Escherichia coli host using fecal DNA from 22 healthy infants and children (most without recent antibiotic exposure), and performed functional selections for resistance to 18 antibiotics from eight drug classes. Resistance-conferring DNA fragments were sequenced (Illumina HiSeq 2000), and reads assembled and annotated with the PARFuMS computational pipeline. Resistance to 14 of the 18 antibiotics was found in stools of infants and children. Recovered genes included chloramphenicol acetyltransferases, drug-resistant dihydrofolate reductases, rRNA methyltransferases, transcriptional regulators, multidrug efflux pumps, and every major class of beta-lactamase, aminoglycoside-modifying enzyme, and tetracycline resistance protein. Many resistance-conferring sequences were mobilizable; some had low identity to any known organism, emphasizing cryptic organisms as potentially important resistance reservoirs. We functionally confirmed three novel resistance genes, including a 16S rRNA methylase conferring aminoglycoside resistance, and two tetracycline-resistance proteins nearly identical to a bifidobacterial MFS transporter (B. longum s. longum JDM301). We provide the first report to our knowledge of resistance to folate-synthesis inhibitors conferred by a predicted Nudix hydrolase (part of the folate synthesis pathway). This functional metagenomic survey of gut-associated resistomes, the largest of its kind to date, demonstrates that fecal resistomes of healthy children are far more diverse than previously suspected, that clinically relevant resistance genes are present even without recent selective antibiotic pressure in the human host, and that cryptic gut microbes are an important resistance reservoir. The observed transferability of gut-associated resistance genes to a gram-negative (E. coli) host also suggests that the potential for gut-associated resistomes to threaten human health by mediating antibiotic resistance in pathogens warrants further investigation.

Induction of the transcription factor Irf8 in the common dendritic cell progenitor (CDP) is required for classical type 1 dendritic cell (cDC1) fate specification, but the mechanisms controlling this induction are unclear. Here we identified Irf8 enhancers via chromatin profiling of DCs and used CRISPR/Cas9 genome editing to assess their roles in Irf8 regulation. An enhancer 32 kilobases downstream of the Irf8 transcriptional start site (+32 kb Irf8 ) that was active in mature cDC1s was required for the development of this lineage, but not for its specification. Instead, a +41 kb Irf8 enhancer previously thought to be active only in plasmacytoid DCs was found to also be transiently accessible in cDC1 progenitors, and deleting this enhancer prevented the induction of Irf8 in CDPs and abolished cDC1 specification. Thus, cryptic activation of the +41 kb Irf8 enhancer in DC progenitors is responsible for cDC1 fate specification.