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.

Figure 1. Antibiotic selections for which resistance was observed.

The percent of libraries from infants (N = 8) and children and adolescents (N = 12) generating colonies resistant to 14 antibiotics is plotted. Two libraries that were <0.1 GB in size were excluded, as they did not have enough genetic diversity to accurately represent the resistance in their source metagenome. Not shown are four antibiotics for which no resistance was found: ciprofloxacin, meropenem, colistin, and cefepime.

Figure 2. Unrooted approximate maximum-likelihood phylogenetic tree constructed using the predicted amino acid sequences of all beta-lactamases in the study set.

Ambler classes are color-coded as indicated in the legend (upper right). At the terminus of each branch, the number of unique amino acid sequences in the terminal cluster is indicated. Branches with identity to extended-spectrum beta-lactamases (ESBLs) are numbered I–V and their putative ESBL classification is listed in the lower left legend of selected genes. A Class D beta-lactamase with high identity to a known OXA-10 is also numbered (VI) and included in the lower left legend. Genes included in the lower left legend that were syntenic with mobile genetic elements are bolded and marked with a §. Clusters of sequences with <55% ID to any known beta-lactamase are marked with a ‡. Nodes with a Shimodaira-Hasegawa (SH) value > = 0.7 are starred. The Shimodaira-Hasegawa score provides a measure of confidence in tree topology, with a maximum score (highest confidence) of 1.0 .

Figure 3. Unrooted approximate maximum-likelihood phylogenetic tree constructed using the predicted amino acid sequences of all aminoglycoside aminotransferases and phosphotransferases in the study set.

Enzyme classes are color-coded as indicated in the legend. At the terminus of each branch, the name of the source contig is listed. Aminoglycoside phosphotransferases that were syntenic with aminoglycoside acetyltransferases are indicated with purple bold text and marked with a ‡. Genes syntenic with a mobile element are bolded and marked with a §. Nodes with a Shimodaira-Hasegawa (SH)-value > = 0.7 are starred.