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Environmental reservoirs of antibiotic resistance are important to human health, and recent evidence indicates that terrestrial resistance reservoirs have expanded during the antibiotic era. Our aim was to study the impact of Cu pollution as a selective driver for the spread of antibiotic resistance in soil. Bacteria were extracted from a well-characterized soil site solely contaminated with CuSO₄ more than 80 years ago and from a corresponding control soil. Pollution-induced bacterial community tolerance (PICT) to Cu and a panel of antibiotics was determined by a novel cultivation-independent approach based on [³H]bromodeoxyuridine (BrdU) incorporation into DNA and by resistance profiling of soil bacterial isolates on solid media. High Cu exposure selected for Cu-tolerant bacterial communities but also coselected for increased community-level tolerance to tetracycline and vancomycin. Cu-resistant isolates showed significantly higher incidence of resistance to five out of seven tested antibiotics (tetracycline, olaquindox, nalidixic acid, chloramphenicol, and ampicillin) than Cu-sensitive isolates. Our BrdU-PICT data demonstrate for the first time that soil Cu exposure coselects for resistance to clinically important antibiotics (e.g., vancomycin) at the bacterial community-level. Our study further indicates that Cu exposure provides a strong selection pressure for the expansion of the soil bacterial resistome.

Toxic metal pollution affects the composition and metal tolerance of soil bacterial communities. However, there is virtually no knowledge concerning the responses of members of specific bacterial taxa (e.g., phyla or classes) to metal toxicity, and contradictory results have been obtained regarding the impact of metals on operational taxonomic unit (OTU) richness. We used tagcoded pyrosequencing of the 16S rRNA gene to elucidate the impacts of copper (Cu) on bacterial community composition and diversity within a well-described Cu gradient (20 to 3,537 g g ؊1 ) stemming from industrial contamination with CuSO 4 more than 85 years ago. DNA sequence information was linked to analysis of pollution-induced community tolerance (PICT) to Cu, as determined by the [ 3 H]leucine incorporation technique, and to chemical characterization of the soil. PICT was significantly correlated to bioavailable Cu, as determined by the results seen with a Cu-specific bioluminescent biosensor strain, demonstrating a specific community response to Cu. The relative abundances of members of several phyla or candidate phyla, including the Proteobacteria, Bacteroidetes, Verrumicrobia, Chloroflexi, WS3, and Planctomycetes, decreased with increasing bioavailable Cu, while members of the dominant phylum, the Actinobacteria, showed no response and members of the Acidobacteria showed a marked increase in abundance. Interestingly, changes in the relative abundances of classes frequently deviated from the responses of the phyla to which they belong. Despite the apparent Cu impacts on Cu resistance and community structure, bioavailable Cu levels did not show any correlation to bacterial OTU richness (97% similarity level). Our report highlights several bacterial taxa responding to Cu and thereby provides new guidelines for future studies aiming to explore the bacterial domain for members of metal-responding taxa.

Copper has been intensively used in industry and agriculture since mid-18(th) century and is currently accumulating in soils. We investigated the diversity of potential active bacteria by 16S rRNA gene transcript amplicon sequencing in a temperate grassland soil subjected to century-long exposure to normal (∼15 mg kg(-1)), high (∼450 mg kg(-1)) or extremely high (∼4500 mg kg(-1)) copper levels. Results showed that bioavailable copper had pronounced impacts on the structure of the transcriptionally active bacterial community, overruling other environmental factors (e.g. season and pH). As copper concentration increased, bacterial richness and evenness were negatively impacted, while distinct communities with an enhanced relative abundance of Nitrospira and Acidobacteria members and a lower representation of Verrucomicrobia, Proteobacteria and Actinobacteria were selected. Our analysis showed the presence of six functional response groups (FRGs), each consisting of bacterial taxa with similar tolerance response to copper. Furthermore, the use of FRGs revealed that specific taxa like the genus Nitrospira and several Acidobacteria groups could accurately predict the copper legacy burden in our system, suggesting a potential promising role as bioindicators of copper contamination in soils.

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Environmental selection of antibiotic resistance may be caused by either antibiotic residues or coselecting agents. Using a strictly controlled experimental design, we compared the ability of metals (Cu or Zn) and tetracycline to (co)select for tetracycline resistance in bacterial communities. Soil microcosms were established by amending agricultural soil with known levels of Cu, Zn, or tetracycline known to represent commonly used metals and antibiotics for pig farming. Soil bacterial growth dynamics and bacterial community-level tetracycline resistance were determined using the [H]leucine incorporation technique, whereas soil Cu, Zn, and tetracycline exposure were quantified by a panel of whole-cell bacterial bioreporters. Tetracycline resistance increased significantly in soils containing environmentally relevant levels of Cu (≥365 mg kg) and Zn (≥264 mg kg) but not in soil spiked with unrealistically high levels of tetracycline (up to 100 mg kg). These observations were consistent with bioreporter data showing that metals remained bioavailable, whereas tetracycline was only transiently bioavailable. Community-level tetracycline resistance was correlated to the initial toxicant-induced inhibition of bacterial growth. In conclusion, our study demonstrates that toxic metals in some cases may exert a stronger selection pressure for environmental selection of resistance to an antibiotic than the specific antibiotic itself.

A two‐part method has been developed for determination of Cd and Zn species in 50‐mL soil solution samples containing low concentrations of Cd and Zn (1–10 μg Cd L−1 and 50–1000 μg Zn L−1). The method uses two cation exchange resins (Amberlite CG 120 and Chelex 100) in a batch‐column‐batch procedure and relies on analytical determinations of Cd and Zn by graphite furnace atomic absorption spectrophotometry. The first part (batch) of the method allows determination of free divalent Cd2+ and Zn2+. This part is experimentally sensitive to cation concentrations and ionic strength and these parameters should be controlled during the experimental procedures. However, it is shown that Cd and Zn concentrations and pH do not influence the method. Speciations performed on samples containing chloride and sulfate were in accordance with theoretical calculations. The second part (column‐batch) of the method operationally separates the complexed fraction into labile complexes, slowly labile complexes, and stable complexes. Chloro complexes were identified as labile complexes, while EDTA complexes were identified as stable complexes. The method works well with relatively small volumes of sample solutions and at low metal concentrations and may be useful in characterization of Cd and Zn in soil solutions.

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