Article Cite This: Environ. Sci. Technol. 2018, 52, 13037−13046
pubs.acs.org/est
Long-Term Effect of Different Fertilization and Cropping Systems on the Soil Antibiotic Resistome Fang Wang,*,†,‡,§,⊥ Min Xu,†,⊥ Robert D. Stedtfeld,∥ Hongjie Sheng,†,‡,§,⊥ Jianbo Fan,† Ming Liu,† Benli Chai,‡,§ Teotonio Soares de Carvalho,‡,§,# Hui Li,‡ Zhongpei Li,† Syed A. Hashsham,‡,§,∥ and James M. Tiedje*,†,‡,§
Environ. Sci. Technol. 2018.52:13037-13046. Downloaded from pubs.acs.org by UNIV OF GOTHENBURG on 12/01/18. For personal use only.
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Key Laboratory of Soil Environment and Pollution Remediation, Institute of Soil Science, Chinese Academy of Sciences, Nanjing 210008, China ‡ Department of Plant, Soil and Microbial Sciences, §Center for Microbial Ecology, ∥Department of Civil and Environmental Engineering, Michigan State University, East Lansing, Michigan 48824, United States ⊥ University of Chinese Academy of Sciences, Beijing 100049, China # Soil Science Department, Federal University of Lavras, Lavras 372000, Brazil S Supporting Information *
ABSTRACT: Different fertilization and cropping systems may influence short- and long-term residues of antibiotic resistance genes (ARGs) and mobile genetic elements (MGEs) in soil. Soils from dryland (peanut) and paddy (rice) fields, which originated from the same nonagricultural land (forested), were treated with either chemical fertilizer, composted manure, or no fertilizer for 26 years before sampling, which occurred one year after the last applications. ARGs and MGEs were investigated using highly parallel qPCR and high-throughput sequencing. Six of the 11 antibiotics measured by LC−MS/MS were detected in the manure applied soil, but not in the nonmanured soils, indicating their source was from previous manure applications. Compared to the unfertilized control, manure application did not show a large accumulation of ARGs in either cropping system but there were some minor effects of soil management on indigenous ARGs. Paddy soil showed higher accumulation of these ARGs, which corresponded to higher microbial biomass than the dryland soil. Chemical fertilizer increased relative abundance of these ARGs in dryland soil but decreased their relative abundance in paddy soil. These results show how long-term common soil management practices affect the abundance and type of ARGs and MGEs in two very different soil environments, one aerobic and the other primarily anaerobic.
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INTRODUCTION Antibiotic resistance has become a global problem as indicated by the worldwide emergence of multidrug-resistant bacteria,1,2 and has led to a call by the United Nations Environment Program (UNEP)3 and the Group of Twenty (G20)4 for international action plans to combat antimicrobial resistance. Wastewater,5,6 sewage,7,8 sludge,9 and manure10−12 are important habitats for antibiotic resistance genes (ARGs). With wastewater irrigation13 and land application of organic wastes (manure and sewage sludge14,15), ARGs-carrying commensal and pathogenic bacteria can spread into arable soils. ARGs can then move on or into crops,16,17 hence posing a potential risk to human health. Soil is a natural host to many ARG-containing bacteria, part of nature’s natural resistome, which was also the original source of many antibiotics. This complex ARG landscape makes it challenging to determine ARG sources and which practices lead to increased or lessened human health risk. © 2018 American Chemical Society
Common agricultural practices like fertilization, irrigation, tillage and different cropping systems are known to or can be expected to affect both the introduced and natural soil resistome. This can be by selection for the antibiotic resistance trait, either by an antibiotic or another function of that trait (coselection), or by selection for other features of populations with the resistance trait being carried along incidentally.18 Manure application is the most likely practice to affect soil ARGs because it often carries large populations of ARGcarrying bacteria, is rich in available carbon for bacterial growth and in recent times may contain antibiotics, metals or other coselectants. For example (Kim et al., 2017;19 Liu et al., 201720) showed that manure from cows regardless of antibiotic Received: Revised: Accepted: Published: 13037
August 3, 2018 October 22, 2018 October 30, 2018 October 30, 2018 DOI: 10.1021/acs.est.8b04330 Environ. Sci. Technol. 2018, 52, 13037−13046
Article
Environmental Science & Technology presence21 influenced levels of ARGs in soil. Long-term application of manure increased sulfonamide resistance genes in the Korea paddy soil.19 The spread of ARGs due to manure application on agricultural fields10 is more attributable to the bacteria it contains than to the nutrients22 although there is some evidence that nitrogen fertilizer has influenced soil ARG content.23 Of the other practices, mineral fertilizer (nitrogen) usage significantly enhanced tetA abundance in a grassland soil,12 but exerted little effect on the resistome in an arable soil.20 However, the NPK application showed only slight or no impact on soil ARGs abundances compared to the no-fertilizer control in a paddy-upland rotation system.24 This discrepancy might be due to the different physicochemical characteristics, cropping system, histories, and native microbiomes of particular dryland and paddy soils. The latter has not been extensively investigated although one long-term study of fertilization practices on sulfonamide resistance gene showed that there is no significant difference between the NPK applied paddy soil and the wetland soil from a mountain site pond in South Korea.19 Applied fertilizer has different fates and effects in the dryland vs paddy soils due to their different water regimes, soil structure and bacterial composition,18,25,26 which should result in different structures and dynamics of the intrinsic and the introduced resistome. However, there are no studies reported on this aspect. Previous studies focused on the short-term effects of different fertilizer treatments on the fate of ARGs in soil20 which generally analyze a smaller subset of ARGs.19 It is evident that more types of ARGs should be considered for a more comprehensive insight into the influence of fertilization and cropping systems on ARGs as well as MGEs in an agricultural environment. Therefore, we used 384 primer sets to investigate ARGs and MGEs composition and abundances in dryland soil and paddy soil that had received chemical fertilizer, composted manure from swine-fed antibiotics, or neither for 26 years in a long-term field experiment. Our objectives were to determine (1) the long-term impact of applying composted manure with history of antibiotic use on the soil ARGs and MGEs profile, (2) the effect of two very different cropping systems, dryland and flooded, and chemical fertilizers on the soil resistome, and (3) to better define the native soil resistome at least in this region.
aerobic cycle. The irrigation water comes from a river nearby the research station. Urea, calcium magnesium phosphate, and potassium chloride were used in CF plots to ensure 112 kg N, 86 kg P, and 147 kg K per ha per yr. The swine manure was from the local farm and composted using windrow composting for 2 months then the compost (Table S1 of the Supporting Information, SI) was applied at 2250 kg dry weight in each MF plot per ha per yr. The antibiotics used by farmer was according to the standard usage practice in China. Antibiotics were widely included in Chinese pig rations from the 1960s through the study period and initially included tetracyclines, tylosin since 199428 and more recently sulfamethoxazole, acetylisovaleryl tylosin and tilmicosin. The same chemical fertilizer was used in the dryland CF plots to ensure 120 kg N, 75 kg P, and 150 kg K per ha per yr and the MF plots received 4500 kg dry weight composted manure per ha per yr.25 The same amount of fertilizers were applied before cultivation each year for 26 years. The soil samples (Table S1) were collected in 2015 just prior to application of the fertilizers, which was April for the peanut field and May for the paddy field; thus, the fields had not received fertilizer for 12 months. This was done to examine the long-term (26 years) effects of fertilizer applications on ARGs in farmland soil. Soils were sampled in plots with about 5 m between different treatments. At each sampling plot, soils were collected from 0 to 20 cm layer at six random locations, mixed to homogenize inside a plastic bag, and immediately frozen on dry ice. The physicochemical properties of soils at sampling sites is listed in Table S1. The composted manure to be applied in the field during 2015 and the irrigation water were also sampled for ARGs analysis. The genomic DNA was extracted from 10 g soil wet weight using the PowerSoil kit (MO BIO, Carlsbad, CA, U.S.A.). The irrigation water (1000 mL) was filtered, and DNA was extracted with a PowerWater Kit (MO BIO, Carlsbad, CA, U.S.A.). The DNA quality and concentration were analyzed with a Qubit Fluorometer (Life Technologies, Eugene, OR, U.S.A.). QPCR Array. To get a comprehensive insight into the influence of fertilizer and cropping systems on ARGs and MGEs in soil, Quantitative PCR reactions with a 384-primer set targeting almost all the known ARGs29,30 were conducted using a Takara (previously Wafergen) SmartChip Real-time PCR system (Fremont, CA, U.S.A.) as reported previously.13 Sample and primers were dispensed into a 5184 well SmartChip using a Multisample Nanodispenser with 100 nl volumes for PCR.13 Each sample was run with three technical replicates. The Wafergen software automatically generated the melting curves and the qPCR Ct values. Initial data processing was performed using R (version 3.1.2).31 Genes detected in only one of the three technical replicates were considered false positives and were removed from the analysis. Negative controls (no added DNA template) were also included. The samples that showed the same amplification with no template controls were excluded from analysis. Some negative (reagent only) controls for the class 1 integron, a marker of fecal pollution, showed a low level of detection therefore we did not include that data in our analyses. However, a strong signal for this gene was detected in the manure and low relative abundance in the manured soil, but not in the chemically fertilized soil. Sequencing of 16S rRNA Gene. A universal 16S rRNA gene primer pair targeting the V4 region was used to measure the total bacterial DNA using qPCR.32 The amplicons were
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MATERIALS AND METHODS Samples and DNA Extraction. Soil samples were collected from a long-term field experiment at the Red Soil Ecological Experimental Station of Chinese Academy of Sciences, located in Yingtan, China. The climate is a subtropical monsoon.25,27 Forest land without human activity was converted to dryland in 1989 to cultivate peanuts, and in 1990 to paddy fields to cultivate rice for a long-term fertilization experiment. Both cropping systems received three treatments: chemical fertilizer (CF), composted swine manure (MF), and no fertilizer (NF). There were three plots (field replicates) for each treatment. Each plot was 30 m2 separated by cement walls to prevent cross-contamination. The crop, fertilization and management was kept constant throughout the 26 years. One peanut crop was grown from April to August with the land fallow for the rest of the year. Two successive rice crops were planted per year covering the April to November time period. The plots were flooded before rice growth but were drained for harvest causing an anaerobic− 13038
DOI: 10.1021/acs.est.8b04330 Environ. Sci. Technol. 2018, 52, 13037−13046
Article
Environmental Science & Technology
Figure 1. Gene copy numbers (per g dry weight or ml) of antibiotic resistance genes (ARGs), mobile genetic elements (MGEs) and 16SrRNA in the composted manure, irrigation water, and soil (n = 3), one-way analysis of variance was conducted among different treatments for ARG, MGE, and 16SrRNA, respectively (a) and the relationship between log-transformed (base 10) gene copy numbers of ARGs and 16S or MGEs in soil (b). DNF, dryland soil with no fertilizer; DCF, dryland soil with chemical fertilizer; DMF, dryland soil with composted manure; WNF, paddy soil with no fertilizer; WCF, paddy soil with chemical fertilizer; and WMF, paddy soil with composted manure.
and finally with anhydrous Na2SO4 (5.0 g) and NaCl (0.5 g). All the samples were centrifuged at 2990g for 10 min followed by pipetting 1.6 mL of the supernatant for further purification using a d-SPE sorbent consisting of C18 (12.5 mg), primary secondary amine (PSA, 12.5 mg), and Na2SO4 (225 mg). The purified sample was analyzed with an LC−MS/MS (Shimadzu, Japan) equipped with a C18 column (150 × 4.6 mm2, particle size: 3 μm, Torrance, CA, U.S.A.) using a binary gradient mobile phase at a flow rate of 350 μL min−1 in which phase A (water) and phase B (acetonitrile) both contained 0.1% (v/v) formic acid as described previously.33 Data Analysis. Multivariate analysis of the difference between profiles of ARGs in different samples was performed in R using the vegan package v2.4−5.34 The Venn map was built in R with the Vennerable package. Canonical correspondence analysis (CCA) between the relative abundance of ARGs and environmental variables (Table S1) was
purified using the AxyPrep DNA Gel Extraction Kit (Axygen Biosciences, Union City, CA, U.S.A.) and quantified using Quanti Fluor-ST (Promega, U.S.A.) followed by combination in equimolar concentrations. Illumina adapters were added by ligation (TruSeq DNA LT Sample Prep Kit) and amplified with 10 cycles for sequencing on an Illumina MiSeq sequencer (San Diego, U.S.A.). Paired-end reads in sample-demultiplexed FASTQ files were (i) merged using RDP Paired-end Reads Assembler32 with the minimum overlap to 10 bases and the minimum read quality score 0.7, p-value
