The shared antibiotic resistome of soil bacteria and human pathogens

Abstract

Soil microbiota represent one of the ancient evolutionary origins of antibiotic resistance and have been proposed as a reservoir of resistance genes available for exchange with clinical pathogens. Using a high-throughput functional metagenomic approach in conjunction with a pipeline for the de novo assembly of short-read sequence data from functional selections (termed PARFuMS), we provide evidence for recent exchange of antibiotic resistance genes between environmental bacteria and clinical pathogens. We describe multidrug-resistant soil bacteria containing resistance cassettes against five classes of antibiotics (β-lactams, aminoglycosides, amphenicols, sulfonamides, and tetracyclines) that have perfect nucleotide identity to genes from diverse human pathogens. This identity encompasses noncoding regions as well as multiple mobilization sequences, offering not only evidence of lateral exchange but also a mechanism by which antibiotic resistance disseminates.

Fig. 1

Functional selection of the AB95 soil metagenomic library with 12 antibiotics (19). (A) Histogram depicting the number of distinct contigs over 500bp recovered from selection with each of the 12 antibiotics. (B) Functional classification of ORFs predicted by PARFuMS, across all selections. (C) Three representative metagenomic fragments; colors match catergorizations depicted in (B). Tick marks represent 300bp and dashed lines indicate common sequence on two distinct fragments. (D) Amino acid identity between antibiotic-resistance ORFs and the closest hit from GenBank, across all selections.

Fig. 2

A gene conferring resistance to D-cycloserine was captured for which sequence was unable to predict resistance function. (A) Resistance-conferring fragment AB95_CY_48 compared to its closest hit from the NCBI nucleotide collection. ORFs of the same color indicate homologous sequence; both nucleotide and amino acid identities are given in shaded regions (nuc/a.a.). Base-pair coordinates flank sequences and each tick mark represents 300bp. (B) Measurements of absorbance at 600nm, taken every 15 minutes, depict growth of E. coli, containing either AB95_CY_48.2 or an empty vector, at clinically-relevant concentrations of D-cycloserine. Measurements are corrected for background absorbance from media-only controls, and are averages of three trials (19).

Fig. 3

Comparison of four AB95-derived resistance fragments to five human pathogenic isolates. The four fragments are depicted along the bottom, and shading indicates high nucleotide identity between the fragments and pathogens (NCBI GenInfo numbers identify each pathogenic isolate). Dark gray shading indicates >99% identity; light grey shading indicates ~88% identity. Base-pair coordinates flank pathogenic sequences and each tick mark represents 800bp. Red ORFs represent resistance genes, yellow represents mobility elements, dark blue represents resistance-associated regulatory elements, and light blue represents other functions.