From Farm to Clinic?: Soil organisms have long been assumed to be an important source of antibiotic resistance genes, in part because of antibiotic-treated livestock and in part because of the natural ecology of antibiotic production in the soil. Forsberg et al. (p. 1107 ) developed a metagenomic protocol to assemble short-read sequence data after antibiotic selection experiments, using 12 different drugs in all antibiotic classes, and compared antibiotic resistance gene sequences between soil bacteria and clinically occurring pathogens. Sixteen sequences, representing seven gene products, were discovered in farmland soil bacteria within long stretches of perfect nucleotide identity with pathogenic proteobacteria.

Functional metagenomic selections for resistance to 18 antibiotics in 18 different soils reveal that bacterial community composition is the primary determinant of soil antibiotic resistance gene content. Antibiotic resistance genes readily move between unrelated bacteria in hospital settings, prompting speculation that the remarkable diversity of resistance genes in soil contributes to an increasing flow of antibiotic resistance from environmental to pathogenic organisms. This study refutes this notion. Kevin Forsberg et al. performed functional metagenomic selections for resistance to 18 antibiotics from a series of agricultural and grassland soils and find that soil bacteria rarely possess the sequence signatures of resistance gene exchange between species. It seems that particular organisms, rather than horizontally exchanged DNA elements, are the major disseminators of antibiotic resistance in the soil. Ancient and diverse antibiotic resistance genes (ARGs) have previously been identified from soil1,2,3, including genes identical to those in human pathogens4. Despite the apparent overlap between soil and clinical resistomes4,5,6, 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 communities7,8,9,10,11,12. However, ARGs are proposed 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 2,895 ARGs we discovered were mostly new, and represent all major resistance mechanisms15. We demonstrate that distinct soil types harbour distinct resistomes, and that the addition of nitrogen fertilizer 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 (genes responsible for horizontal gene transfer between bacteria such as transposases and integrases) syntenic with ARGs were rare in soil by comparison with sequenced pathogens, suggesting that ARGs may not transfer between soil bacteria as readily as is observed between human pathogens. 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.

Methods for profiling DNA methylation differ in the physical principles used to detect modified cytosines. Harris et al. compare the performances of four sequencing-based technologies for genome-wide analysis of DNA methylation and combine two methods to enable detection of allelic differences in epigenetic marks. Analysis of DNA methylation patterns relies increasingly on sequencing-based profiling methods. The four most frequently used sequencing-based technologies are the bisulfite-based methods MethylC-seq and reduced representation bisulfite sequencing (RRBS), and the enrichment-based techniques methylated DNA immunoprecipitation sequencing (MeDIP-seq) and methylated DNA binding domain sequencing (MBD-seq). We applied all four methods to biological replicates of human embryonic stem cells to assess their genome-wide CpG coverage, resolution, cost, concordance and the influence of CpG density and genomic context. The methylation levels assessed by the two bisulfite methods were concordant (their difference did not exceed a given threshold) for 82% for CpGs and 99% of the non-CpG cytosines. Using binary methylation calls, the two enrichment methods were 99% concordant and regions assessed by all four methods were 97% concordant. We combined MeDIP-seq with methylation-sensitive restriction enzyme (MRE-seq) sequencing for comprehensive methylome coverage at lower cost. This, along with RNA-seq and ChIP-seq of the ES cells enabled us to detect regions with allele-specific epigenetic states, identifying most known imprinted regions and new loci with monoallelic epigenetic marks and monoallelic expression.

Antibiotic resistance is a dire clinical problem with important ecological dimensions. While antibiotic resistance in human pathogens continues to rise at alarming rates, the impact of environmental resistance on human health is still unclear. To investigate the relationship between human-associated and environmental resistomes, we analyzed functional metagenomic selections for resistance against 18 clinically relevant antibiotics from soil and human gut microbiota as well as a set of multidrug-resistant cultured soil isolates. These analyses were enabled by Resfams, a new curated database of protein families and associated highly precise and accurate profile hidden Markov models, confirmed for antibiotic resistance function and organized by ontology. We demonstrate that the antibiotic resistance functions that give rise to the resistance profiles observed in environmental and human-associated microbial communities significantly differ between ecologies. Antibiotic resistance functions that most discriminate between ecologies provide resistance to β-lactams and tetracyclines, two of the most widely used classes of antibiotics in the clinic and agriculture. We also analyzed the antibiotic resistance gene composition of over 6000 sequenced microbial genomes, revealing significant enrichment of resistance functions by both ecology and phylogeny. Together, our results indicate that environmental and human-associated microbial communities harbor distinct resistance genes, suggesting that antibiotic resistance functions are largely constrained by ecology.

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.