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Comparison of metals and tetracycline as selective agents for development of tetracycline resistant bacterial communities in agricultural soil Jianxiao Song, Christopher Rensing, Peter E. Holm, Marko P.J. Virta, and Kristian Koefoed Brandt Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.6b05342 • Publication Date (Web): 15 Feb 2017 Downloaded from http://pubs.acs.org on February 16, 2017
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Comparison of metals and tetracycline as selective
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agents for development of tetracycline resistant
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bacterial communities in agricultural soil
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Jianxiao Song,† Christopher Rensing,‡ Peter E. Holm,† Marko Virta,§ and Kristian K. Brandt*,† †
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Copenhagen, 1871 Frederiksberg, Denmark ‡
College of Natural Resources and the Environment, Fujian Agriculture and Forestry University,
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Department of Plant and Environmental Sciences, Faculty of Science, University of
350002 Fuzhou, China §
Department of Food and Environmental Sciences, University of Helsinki, PL 56 Helsinki, Finland
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KEYWORDS: Antibiotic resistance, copper, environmental selection, tetracycline, zinc
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ABSTRACT
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Environmental selection of antibiotic resistance may be caused by either antibiotic residues or by
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co-selecting agents. Using a strictly controlled experimental design we compared the ability of
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metals (Cu or Zn) and tetracycline to (co)select for tetracycline resistance in bacterial
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communities. Soil microcosms were established by amending agricultural soil with known levels
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of Cu, Zn or tetracycline known to represent commonly used metals and antibiotics for pig
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farming. Soil bacterial growth dynamics and bacterial community-level tetracycline resistance
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were determined using the [3H]leucine incorporation technique, whereas soil Cu, Zn, and
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tetracycline exposure were quantified by a panel of whole-cell bacterial bioreporters.
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Tetracycline resistance increased significantly in soils containing environmentally relevant levels
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of Cu (≥365 mg kg-1) and Zn (≥264 mg kg-1), but not in soil spiked with unrealistically high
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levels of tetracycline (up to 100 mg kg-1). These observations were consistent with bioreporter
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data showing that metals remained bioavailable, whereas tetracycline was only transiently
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bioavailable. Community-level tetracycline resistance was correlated to the initial toxicant-
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induced inhibition of bacterial growth. In conclusion, our study demonstrates that toxic metals in
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some cases may exert a stronger selection pressure for environmental selection of resistance to
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an antibiotic than the specific antibiotic itself.
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Introduction
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The ongoing dissemination of antibiotic resistance in pathogenic bacteria constitutes a global
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challenge to mankind and an environmental dimension to the antibiotic resistance crisis is
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increasingly being recognized.1–3 In this regard, agricultural soils are important as they provide
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rich sources of novel types of antibiotic resistance determinants yet-to-be recruited by human
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pathogens. Hence, antibiotic resistance genes (ARGs) are common in soil that is known to harbor
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a plethora of antibiotic producers and degraders4,5 and extensive transfer of ARGs between
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bacteria in soil and pathogenic bacteria in humans has been indicated.6,7
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There is growing evidence that the human use of antibiotics has caused a vast enrichment of
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ARGs in agricultural soils.8–10 Less is known about the relative importance of the underlying
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processes responsible for this expansion of the soil bacterial resistome. These processes may
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include dispersal of antibiotic resistant bacteria or ARGs from animals to soil via manure,
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horizontal gene transfer, and environmental selection of antibiotic resistance caused by antibiotic
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residues or other selecting agents such as toxic metals that can co-select for antibiotic
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resistance.2,8 Various co-selection mechanisms (i.e. co-resistance, cross-resistance and co-
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regulation) have been known for decades, but only little is known about their relative importance
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in soil.11
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In order to perform a human health risk assessment for the environmental development and
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transfer of antibiotic resistance in agricultural soil it will be needed to distinguish the individual
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factors responsible for the dissemination of antibiotic resistance.2 This implies that we need to
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predict the degree to which selection of antibiotic resistance is taking place in agricultural soils
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and to identify the selective agents. Widespread selection of antibiotic resistance above
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background levels in agricultural soils requires that antibiotic resistant bacteria have a selective
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advantage over antibiotic susceptible bacteria; in other words, a selection pressure must be
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present. Both antibiotics and metals are used in animal farming, where copper (Cu) and zinc (Zn)
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are widely used to complement antibiotics in order to promote animal growth and alleviate post-
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weaning diarrhea.12 Manure-amended soils will thus contain elevated levels not only of antibiotic
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residues, but also of Cu and Zn making it relevant to compare their abilities to select for
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antibiotic resistance. As opposed to antibiotics, Cu and Zn are elements and thus persistent in
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soil where they consequently accumulate in agricultural soils receiving animal manure.13 These
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metals may also accumulate to toxic levels due to use of Cu-based pesticides or industrial
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pollution.14–16 By contrast, antibiotics probably only rarely accumulate to toxic levels in
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agricultural soils17,18 and metals are thus more likely to exert toxic effects in agricultural soils
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than antibiotics are.
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To the best of our knowledge, no previous controlled studies have directly compared the
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ability of metals and antibiotics to (co)-select for antibiotic resistance. Hence, we aimed to
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directly compare the abilities of Cu, Zn, and tetracycline to (co)-select for tetracycline resistance
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in soil bacterial communities operationally defined as an increased ability of extracted soil
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bacteria to grow in the presence of tetracycline. To this end, we performed strictly controlled
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experiments employing soil microcosms spiked with known amounts of Cu, Zn or tetracycline.
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Importantly, manure was not included as part of the experimental design in an attempt to fully
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resolve causal relationships. Tetracycline was chosen as the model antibiotic as tetracyclines
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represent one of the most commonly used classes of veterinary antibiotics19 and as it is listed by
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the WHO as a critically important antibiotic.20 Comparison of selection pressures for tetracycline
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resistance was carried out by a cultivation-independent pollution-induced community tolerance
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(PICT) approach with broad bacterial community coverage and no requirement for a priori
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knowledge on resistance mechanisms in individual members of the soil bacterial community.
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PICT analysis was performed after a selection phase of 9 weeks and was complemented by
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studies of in situ bacterial growth dynamics and determination of Cu, Zn, and tetracycline
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bioavailability over time using an array of whole-cell bacterial bioreporters.
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Materials and Methods
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Soil sampling and experimental set-up. An agricultural soil (sandy loam) was
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representatively sampled by multiple coring in February, 2014, Taastrup, Denmark from the
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unfertilized control plots of a field experiment that was initiated in 2002 at an experimental farm
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of the University of Copenhagen. Detailed physicochemical and microbiological soil
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characterization are available from multiple investigations of the same soil plots.21–23 Total Cu
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and Zn contents were 15 mg kg-1 and 57 mg kg-1, respectively. After 4 weeks of storage at 4°C,
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the soil was air-dried for 5 hours at 22 °C, sieved (2 mm mesh size), and pre-incubated (5 days)
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at 15 °C in the dark. Replicated microcosms (n=3) were subsequently set up with 50 g (dry wt)
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soil in 324-ml serum vials sealed with Para-film. Different concentrations of CuSO4 (CAS no:
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7758-99-8, Merck), ZnSO4 (CAS no: 7446-20-0, Merck) or tetracycline (tetracycline
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hydrochloride, CAS no: 64-75-5, Sigma) were spiked to soil by pipetting droplets (2.5 mL)
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resulting in a final soil moisture of 16.0% (dry wt.). Soils were repeatedly gently mixed during
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the spiking process to ensure homogeneous distribution of the contaminants. Using freshly
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prepared solutions, tetracycline was amended to soil to increase nominal concentrations by 0, 1,
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10, or 100 mg kg-1 dry soil. Cu was added to increase total soil Cu concentrations by 0, 33.3,
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100, 333 or 1000 mg kg-1 dry soil, whereas Zn was added to increase total soil Zn concentrations
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by 0, 165, 500, 1650 or 5000 mg kg-1 dry soil. Total soil Cu and Zn concentrations were
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quantified by atomic absorption spectroscopy (AAS PerkinElmer PinAAcle 500) for dose
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confirmation (see supporting information; Table S1 for dose confirmation). All soil microcosms
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were incubated at 15°C in the dark and sampled (see below for quantities used) after 3 hours, 1
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day, 1 week, 4 weeks and 9 weeks for determination of bacterial growth dynamics and
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bioavailable Cu, Zn or tetracycline (see below).
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Detection of pollution-induced community tolerance (PICT). Environmental selection
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pressures for development of tetracycline resistant bacterial communities was determined by a
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cultivation-independent PICT approach based on the use of the [3H]leucine incorporation
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technique24 for PICT detection. PICT detection was performed after 9 weeks of microcosm
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incubation as described previously.22 In brief, soil bacterial communities were extracted with
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MES buffer (CAS no.4432-31-9, Sigma, 4-morpholineethanesulfonic acid, 20 mM, pH=6.4)
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using a soil: water ratio of 1:10 (w/v) and 1.5 mL aliquots of soil bacterial suspensions were pre-
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incubated at various concentrations (0, 0.25, 1, 4, 8, 16, 32 or 64 mg L-1) of tetracycline (n = 3)
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for 30 min at 22°C. Incubations were subsequently started by addition of [3H]Leucine (2.59 TBq
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mmol-1, 35 MBq ml-1, Amersham, Hillerød) and nonradiolabled leucine to give a final
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radioactivity of 6kBq per microtube and leucine concentration of 200 nM and terminated after 2
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h by addition of trichloroacetic acid (TCA). Tetracycline dose-response curves for bacterial
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community tolerance were fitted using the four parameter logistic function in the drc-package in
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R version 3.0.1. Bacterial community tolerance was also quantified as a tolerance index (TI) for
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each soil microcosm: TI = Linc_TC/Linc-control, where Linc-control equals the average [3H]leucine
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incorporation rate in replicated soil bacterial suspensions amended with Milli-Q water (absence
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of tetracycline) and Linc_TC equals the average [3H]leucine incorporation rate in replicated soil
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bacterial suspensions amended with tetracycline (0.25, 1 or 4 mg L-1). Threshold concentrations
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were selected based on complete dose-response data.
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Determination of bacterial growth rates. Soil bacterial growth in each microcosm was
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measured by the [3H]leucine incorporation technique.24 In brief, soil bacteria were extracted from
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1 g of moist soil with 10 mL of Milli-Q water and relative [3H]leucine incorporation rates were
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measured as described previously.25 All growth rates were normalized relative to the growth rate
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in control soil after 3 hours of incubation.
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Bioreporter analysis of bioavailable Cu, Zn and tetracycline in spiked soil. Bioavailable
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Cu, Zn and tetracycline were measured in aqueous soil extracts which were derived from soil
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microcosm as described previously.26 In brief, 1 g of moist soil was taken from each microcosm
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and mixed with 5 mL Milli-Q water in a 15-mL Falcon tube (polypropylene)
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hours of shaking on a horizontal shaker (250 rpm, 22 °C). The supernatant was collected after
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centrifugation (10000 g, 22 °C and 10 min) and stored at -20 °C for subsequent bioreporter
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analysis.
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, followed by 2
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A panel of whole-cell bacterial bioreporters (bioreporters) was used. Bioavailable Cu was
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determined using Pseudomonas fluorescens strain DF57-Cu15, which carries a chromosomal
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insertion of a promoter-less Tn5::luxAB cassette controlled by a Cu-inducible promoter.28
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Bioavailable Zn was determined using a Pseudomonas putida KT2440 strain transformed with
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the sensor plasmid pDNPczc1lux, which contains the luxCDABE operon from Photorhabdus
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luminescens under the control of czcCBA1 promoter from P. putida.29 Bioavailable tetracycline
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was determined using Escherichia coli K12 (pTetLux1) carrying the luxCDABE operon on a
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sensor plasmid under the control of the tetA promoter from pASK75.30,31 Bioavailable Cu
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([Cu]bio), Zn ([Zn]bio) or tetracycline ([TC]bio) were operationally defined as water-extractable
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Cu, Zn or tetracycline species that were able to induce reporter gene expression in bioreporter
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cells. Exponential phase bioreporter cells were harvested and mixed with an equal volume soil-
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water extracts (see details on strain preparation and bioreporter assay in supporting information)
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before quantification of bioluminescence in a BMG FluoStar Optima plate reader (BMG
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Labtech, Offenburg, Germany). [Cu]bio was calculated as described previously assuming zero
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bioavailability of particle associated Cu,32 whereas [Zn]bio and [TC]bio were determined
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essentially as described previously with full details given as supporting information.
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Statistical analyses. SigmaPlot Version 12.5 (Systat Software, Point Richmond, CA) was
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used for correlation test and significance testing of treatments effects. Nonlinear correlations
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between bacterial community tolerance indices and bacterial growth rates were analyzed with the
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nonparametric Spearman rank correlation test. Significance testing of treatment effects on
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bacterial growth and bacterial community tolerance indices was performed by one-way ANOVA
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and all multiple pairwise comparisons by the Holm-Sidak method. When required, raw data was
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log transformed. The dose-response curves for bacterial community tolerance were fitted with
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four parameter logistic function with the drc-package in R version 3.0.1.
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Results
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Bacterial community tolerance to tetracycline induced by metals and tetracycline. Cu and
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Zn co-selected for tetracycline resistant bacterial communities in a dose-dependent manner in
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soil microcosms during the 9-week long PICT selection phase. Hence, increased community
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tolerance to tetracycline was observed at high Cu (≥333 mg kg-1, Figure 1A) and Zn levels (≥500
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mg kg-1, Figure 1B). Half-maximal effective concentrations (EC50 values) could not be reliably
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determined from the dose-response curves due to insufficient inhibition of growth (i.e. due to
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high community tolerance) observed for high metal treatments. Community tolerance to
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tetracycline was therefore quantified as a tolerance index (TI) using a threshold concentration of
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1 mg L-1 for tolerance determination (Figure 1D and 1E). Tolerance indices (TIs) increased
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progressively with elevated metal (Cu or Zn) exposure during the 9-weeks selection phase and
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significantly increased community tolerance to tetracycline was observed at high Cu (≥365 mg
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kg-1, n=3, p< 0.01, Figure 1D; Table S1) and Zn levels (≥488 mg kg-1, n=3, p
