Pediatric Fecal Microbiota Harbor Diverse and Novel Antibiotic Resistance Genes

Abstract

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.

Metagenomic Approach for Identification of the Pathogens Associated with Diarrhea in Stool Specimens

“…tet genes are the most common resistance genes identified in stool samples from healthy adults (34,35). Indeed, a recent study indicated that tetracycline, beta-lactamases, and multiple drug resistance genes were commonly found in the stool of children Ͻ12 months of age (36). We also identified genes encoding multidrug efflux system proteins in one sample.…”

Improved annotation of antibiotic resistance determinants reveals microbial resistomes cluster by ecology

“…All functional metagenomic selections in this analysis were selected, sequenced and assembled into contigs as previously described (Forsberg et al, 2012(Forsberg et al, , 2014Moore et al, 2013). Briefly, metagenomic plasmid libraries prepared in Escherichia coli DH10B host were selected for resistant inserts on Luria-Bertani or Mueller-Hinton agar plates containing kanamycin (50 mg ml À 1 ; plasmid resistance marker) plus the antibiotic of interest at a concentration toxic to wild-type E. coli host.…”

The Prehistory of Antibiotic Resistance

“…To that end, novel techniques like single-cell sequencing and metagenomic analyses (the analysis of all DNA isolated directly from an environment without prior culture) are improving access to branches on the tree of life that have been previously undersampled (Jansson and Tas 2014). A recent series of analyses by the Dantas laboratory using culture-independent techniques have yielded an enormous wealth of resistance gene sequence data that will help in the phylogenetics and dating of resistance (Forsberg et al 2012(Forsberg et al , 2014Moore et al 2013;Pehrsson et al 2013;Gibson et al 2015). These studies have used "functional metagenomics," in which the entire microbial metagenome is cloned into an expression vector, transformed into an expression host (Escherichia coli) and selected on an antibiotic of interest.…”