Functional characterization of bacteria isolated from ancient arctic soil exposes diverse resistance mechanisms to modern antibiotics

Abstract

Using functional metagenomics to study the resistomes of bacterial communities isolated from different layers of the Canadian high Arctic permafrost, we show that microbial communities harbored diverse resistance mechanisms at least 5,000 years ago. Among bacteria sampled from the ancient layers of a permafrost core, we isolated eight genes conferring clinical levels of resistance against aminoglycoside, β-lactam and tetracycline antibiotics that are naturally produced by microorganisms. Among these resistance genes, four also conferred resistance against amikacin, a modern semi-synthetic antibiotic that does not naturally occur in microorganisms. In bacteria sampled from the overlaying active layer, we isolated ten different genes conferring resistance to all six antibiotics tested in this study, including aminoglycoside, β-lactam and tetracycline variants that are naturally produced by microorganisms as well as semi-synthetic variants produced in the laboratory. On average, we found that resistance genes found in permafrost bacteria conferred lower levels of resistance against clinically relevant antibiotics than resistance genes sampled from the active layer. Our results demonstrate that antibiotic resistance genes were functionally diverse prior to the anthropogenic use of antibiotics, contributing to the evolution of natural reservoirs of resistance genes.

Fig 1. Distribution of antibiotic resistance genes isolated from ancient permafrost bacteria and its overlaying active layer.

A) Samples were collected from Eureka on Ellesmere Island, Canada. B) Antibiotic resistance genes isolated using functional metagenomics were traced back to bacterial strains isolated at different depths of a single 14-m core. C) Resistance genes were mapped onto the 16S rRNA gene phylogeny of all ancient (red) and modern (black) bacterial isolates used in this study. Each resistance gene is represented by a unique color and shape combination: resistance to β-lactams (red), tetracyclines (green), and aminoglycosides (blue) as explained in Fig. 2. We could not identify the host strain for two resistance genes, most likely because we could not revive three ancient strains.

Fig 2. List of resistance genes and their predicted annotation.

Fig 3. Nucleotide similarities of resistance genes isolated from ancient (red) and modern (black) bacteria with the closest homologous genes found in A) any bacteria or in B) a pathogenic bacteria.

Fig 4. Resistance (A-C) and cross-resistance (D-F) levels of resistance genes isolated from ancient permafrost and its overlaying active layer.

Each unique gene is depicted by a shape and color combination based on sampling site and antibiotic on which it was isolated (shown on top of panels): A) & D) β-lactams, penicillin (PEN) & carbenicillin (CAR); B) & E) tetracyclines, tetracycline (TET) & doxycycline (DOX); and C) & F) aminoglycosides, sisomicin (SIS) & amikacin (AMK). In panels A) to C), each point shows resistance to antibiotics indicated at left (measured as minimum inhibitory concentration, MIC). Grey panels indicate resistance levels to the drug in which genes were isolated, and white panels show cross-resistance to the other drug in the same class. Dashed line indicates MIC of control libraries. Panels D) to F) show slopegraphs of cross-resistance between antibiotics of a same family. The left axis represents relative resistance (MIC of the isolated genes / MIC of the control E. coli library) in the antibiotics where the gene was isolated. The right axis represents the relative fitness of the genes in the other antibiotic of the same class. Any slope that doesn’t go down to one on the right axis indicates some degree of cross-resistance.