Long-Term Effect of Different Fertilization and Cropping Systems on

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Characterization of Natural and Affected Environments

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, Teotônio Soares de Carvalho, Hui Li, Zhongpei Li, Syed A. Hashsham, and James M. Tiedje Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.8b04330 • Publication Date (Web): 30 Oct 2018 Downloaded from http://pubs.acs.org on October 31, 2018

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Environmental Science & Technology

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Long-term Effect of Different Fertilization and Cropping Systems on

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the Soil Antibiotic Resistome

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Fang Wang†,‡,§,◊,∗, Min Xu†,◊, Robert D. Stedtfeld┴, Hongjie Sheng†,‡,§,◊, Jianbo Fan†,

4

Ming Liu†, Benli Chai‡,§, Teotonio Soares de Carvalho‡,§, ǁǁ, Hui Li‡, Zhongpei Li†, Syed

5

A. Hashsham‡,§,┴, and James M. Tiedje†, ‡, §,∗

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†

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Science, Chinese Academy of Sciences, Nanjing 210008, China

8

‡

9 10 11 12

Key Laboratory of Soil Environment and Pollution Remediation, Institute of Soil

Department of Plant, Soil and Microbial Sciences, § Center for Microbial Ecology,

┴

Department of Civil and Environmental Engineering, Michigan State University, MI

48824, USA ◊ ǁ‖

University of Chinese Academy of Sciences, Beijing 100049, China Soil Science Department, Federal University of Lavras, Lavras 372000, Brazil

13 14

*

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[email protected].

Corresponding authors. Fang Wang, [email protected]; James M. Tiedje,

1

ACS Paragon Plus Environment


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Table of Contents (TOC)

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ABSTRACT

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Different fertilization and cropping systems may influence short- and long-term

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residues of antibiotic resistance genes (ARGs) and mobile genetic elements (MGEs) in

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soil. Soils from dryland (peanut) and paddy (rice) fields, which originated from the

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same non-agricultural land (forested), were treated with either chemical fertilizer,

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composted manure, or no fertilizer for 26 years before sampling, which occurred one

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year after the last applications. ARGs and MGEs were investigated using highly parallel

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qPCR and high-throughput sequencing. Six of the eleven antibiotics measured by LC-

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MS-MS were detected in the manure applied soil, but not in the non-manured soils,

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indicating their source was from previous manure applications. Compared to the

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unfertilized control, manure application did not show a large accumulation of ARGs in

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either cropping system but there were some minor effects of soil management on

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indigenous ARGs. Paddy soil showed higher accumulation of these ARGs, which

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corresponded to higher microbial biomass than the dryland soil. Chemical fertilizer

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increased relative abundance of these ARGs in dryland soil but decreased their relative

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abundance in paddy soil. These results show how long-term common soil management

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practices affect the abundance and type of ARGs and MGEs in two very different soil

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environments, one aerobic and the other primarily anaerobic.

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INTRODUCTION

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Antibiotic resistance has become a global problem as indicated by the world-wide

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emergence of multidrug-resistant bacteria, 1, 2 and has led to a call by the United Nations

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Environment Program (UNEP) 3 and the Group of Twenty (G20)

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action plans to combat antimicrobial resistance. Wastewater,

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and manure

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wastewater irrigation

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sludge 14,15), ARGs-carrying commensal and pathogenic bacteria can spread into arable

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soils. ARGs can then move on or into crops 16,17, hence posing a potential risk to human

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health.

10-12

5, 6

4

for international

sewage,

7, 8

sludge

9

are important habitats for antibiotic resistance genes (ARGs). With 13

and land application of organic wastes (manure and sewage

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Soil is also naturally host to many ARG-containing bacteria, part of nature’s

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natural resistome, which was also the original source of many antibiotics. This complex

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ARG landscape makes it challenging to determine ARG sources and which practices

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lead to increased or lessened human health risk. Common agricultural practices like

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fertilization, irrigation, tillage and different cropping systems are known to or can be

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expected to affect both the introduced and natural soil resistome. This can be by

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selection for the antibiotic resistance trait, either by an antibiotic or another function of

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that trait (co-selection), or by selection for other features of populations with the

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resistance trait being carried along incidentally. 18 Manure application is the most likely

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practice to affect soil ARGs because it often carries large populations of ARG-carrying

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bacteria, is rich in available carbon for bacterial growth and in recent times may contain

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antibiotics, metals or other co-selectants. For example (Kim et al., 2017 19; Liu et al.,

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2017 20) showed that manure from cows regardless of antibiotic presence 21 influenced

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levels of ARGs in soil. Long-term application of manure increased sulfonamide

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resistance genes in the Korea paddy soil

19

. The spread of ARGs due to manure

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application on agricultural fields 10 is more attributable to the bacteria it contains than

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to the nutrients 22 although there is some evidence that nitrogen fertilizer has influenced

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soil ARG content. 23

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Of the other practices, mineral fertilizer (nitrogen) usage significantly enhanced 12

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tetA abundance in a grassland soil,

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arable soil. 20 However, the NPK application showed only slight or no impact on soil

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ARGs abundances compared to the no-fertilizer control in a paddy-upland rotation

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system.

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characteristics, cropping system, histories and native microbiomes of particular dryland

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and paddy soils. The latter has not been extensively investigated although one long-

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term study of fertilization practices on sulfonamide resistance gene showed that there

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is no significant difference between the NPK applied paddy soil and the wetland soil

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from a mountain site pond in South Korea 19. Applied fertilizer has different fates and

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effects in the dryland vs. paddy soils due to their different water regimes, soil structure

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and bacterial composition,

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dynamics of the intrinsic and the introduced resistome. However, there are no studies

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reported on this aspect.

24

but exerted little effect on the resistome in an

This discrepancy might be due to the different physico-chemical

18, 25-26

which should result in different structures and

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Previous studies focused on the short-term effects of different fertilizer treatments

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on the fate of ARGs in soil 20 which generally analyze a smaller subset of ARGs 19. It

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is evident that more types of ARGs should be considered for a more comprehensive

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insight into the influence of fertilization and cropping systems on ARGs as well as

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MGEs in an agricultural environment. Therefore, we used 384 primer sets to investigate

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ARGs and MGEs composition and abundances in dryland soil and paddy soil that had

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received chemical fertilizer, composted manure from swine-fed antibiotics, or neither

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for 26 years in a long-term field experiment. Our objectives were to determine 1) the 5



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long-term impact of applying composted manure with history of antibiotic use on the

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soil ARGs and MGEs profile, 2) the effect of two very different cropping systems,

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dryland and flooded, and chemical fertilizers on the soil resistome, and 3) to better

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define the native soil resistome at least in this region.

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MATERIALS AND METHODS

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Samples and DNA Extraction. Soil samples were collected from a long-term field

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experiment at the Red Soil Ecological Experimental Station of Chinese Academy of

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Sciences, located in Yingtan, China. The climate is a subtropical monsoon. 25, 27 Forest

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land without human activity was converted to dryland in 1989 to cultivate peanuts, and

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in 1990 to paddy fields to cultivate rice for a long-term fertilization experiment. Both

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cropping systems received three treatments: chemical fertilizer (CF), composted swine

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manure (MF), and no fertilizer (NF). There were three plots (field replicates) for each

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treatment. Each plot was 30 m2 separated by cement walls to prevent cross-

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contamination. The crop, fertilization and management was kept constant throughout

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the 26 years. One peanut crop was grown from April to August with the land fallow for

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the rest of the year. Two successive rice crops were planted per year covering the April

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to November time period. The plots were flooded before rice growth but were drained

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for harvest causing an anaerobic-aerobic cycle. The irrigation water comes from a river

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nearby the research station. Urea, calcium magnesium phosphate and potassium

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chloride were used in CF plots to ensure 112 kg N, 86 kg P and 147 kg K per ha per yr.

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The swine manure was from the local farm and composted using windrow composting

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for 2 months then the compost (Table S1) was applied at 2,250 kg dry weight in each

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MF plot per ha per yr. The antibiotics used by farmer was according to the standard

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usage practice in China. Antibiotics were widely included in Chinese pig rations from 6


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the 1960s through the study period and initially included tetracyclines, tylosin since

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1994

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The same chemical fertilizer was used in the dryland CF plots to ensure 120 kg N, 75

114

kg P and 150 kg K per ha per yr and the MF plots received 4,500 kg dry weight

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composted manure per ha per yr. 25 The same amount of fertilizers were applied before

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cultivation each year for 26 years. The soil samples (Table S1) were collected in 2015

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just prior to application of the fertilizers, which was April for the peanut field and May

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for the paddy field; thus, the fields had not received fertilizer for 12 months. This was

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done to examine the long-term (26 years) effects of fertilizer applications on ARGs in

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farmland soil. Soils were sampled in plots with about 5 m between different treatments.

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At each sampling plot, soils were collected from 0-20 cm layer at six random locations,

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mixed to homogenize inside a plastic bag, and immediately frozen on dry ice. The

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physicochemical properties of soils at sampling sites is listed in Table S1. The

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composted manure to be applied in the field during 2015 and the irrigation water were

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also sampled for ARGs analysis. The genomic DNA was extracted from 10 g soil wet

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weight using the PowerSoil® kit (MO BIO, Carlsbad, CA, USA). The irrigation water

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(1000 ml) was filtered, and DNA was extracted with a PowerWater® Kit (MO BIO,

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Carlsbad, CA, USA). The DNA quality and concentration were analyzed with a Qubit

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Fluorometer (Life Technologies, Eugene, OR, USA).

28

and more recently sulfamethoxazole, acetylisovaleryl tylosin and tilmicosin.

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QPCR Array. To get a comprehensive insight into the influence of fertilizer and

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cropping systems on ARGs and MGEs in soil, Quantitative PCR reactions with a 384-

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primer set targeting almost all the known ARGs

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(previously Wafergen) SmartChip Real-time PCR system (Fremont, CA, USA) as

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reported previously. 13 Sample and primers were dispensed into a 5,184 well SmartChip

29, 30

were conducted using a Takara

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using a Multi-sample Nanodispenser with 100 nl volumes for PCR. 13 Each sample was

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run with three technical replicates. The Wafergen software automatically generated the

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melting curves and the qPCR Ct values. Initial data processing was performed using R

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(version 3.1.2).

140

considered false positives and were removed from the analysis. Negative controls (no

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added DNA template) were also included. The samples that showed the same

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amplification with no template controls were excluded from analysis. Some negative

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(reagent only) controls for the class 1 integron, a marker of fecal pollution, showed a

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low level of detection therefore we did not include that data in our analyses. However

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a strong signal for this gene was detected in the manure and low relative abundance in

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the manured soil, but not in the chemically fertilized soil.

31

Genes detected in only one of the three technical replicates were

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Sequencing of 16S rRNA Gene. A universal 16S rRNA gene primer pair targeting the

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V4 region was used to measure the total bacterial DNA using qPCR. 32 The amplicons

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were purified using the AxyPrep DNA Gel Extraction Kit (Axygen Biosciences, Union

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City, CA, USA) and quantified using Quanti Fluor™-ST (Promega, USA) followed by

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combination in equimolar concentrations. Illumina adapters were added by ligation

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(TruSeq DNA LT Sample Prep Kit) and amplified with 10 cycles for sequencing on an

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Illumina MiSeq sequencer (San Diego, USA). Paired-end reads in sample-

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demultiplexed FASTQ files were (i) merged using RDP Paired-end Reads Assembler 32

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with the minimum overlap to 10 bases and the minimum read quality score < 25. The

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phylogenetic affiliation of 16S rRNA gene sequence was determined using the RDP

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Classifier at a confidence threshold of 90%.

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Analysis of Antibiotics in Soil. Antibiotics in soil samples were analyzed using LC8


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MS/MS after extraction and purification as described previously.

Briefly, 2.0 g soil

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was vortexed with Na 2 EDTA solution (2.0 ml x 150 mg l-1) and mixed further with 5

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ml acetonitrile/methanol (65/35, v/v), and finally with anhydrous Na 2 SO 4 (5.0 g) and

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NaCl (0.5 g). All the samples were centrifuged at 2,990 g for 10 min followed by

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pipetting 1.6 ml of the supernatant for further purification using a d-SPE sorbent

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consisting of C 18 (12.5 mg), primary secondary amine (PSA, 12.5 mg) and Na 2 SO 4

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(225 mg). The purified sample was analyzed with an LC-MS/MS (Shimadzu, Japan)

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equipped with a C 18 column (150 mm×4.6 mm, particle size: 3 µm, Torrance, CA, USA)

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using a binary gradient mobile phase at a flow rate of 350 µl min-1 in which phase A

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(water) and phase B (acetonitrile) both contained 0.1% (v/v) formic acid as described

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previously. 33

172 173

Data Analysis. Multivariate analysis of the difference between profiles of ARGs in

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different samples was performed in R using the vegan package v2.4-5. 34 The Venn map

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was built in R with the Vennerable package. Canonical correspondence analysis (CCA)

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between the relative abundance of ARGs and environmental variables (Table S1) was

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performed followed by the Mantel test using the vegan package. Environmental

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variables were chosen based on significance calculated from individual CCA results

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and variance inflation factors calculated during CCA. Fold of change (FC) was used to

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compare relative abundance of ARGs between different treatments: ΔCt = Ct

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Ct (16S), ΔΔCt = Ct (target) - Ct (reference), FC = 2-∆∆Ct, where Ct is the threshold cycle. The

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reference sample – Ct

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fertilization or of cropping system. Network analysis based on Spearman correlation 35

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was performed to represent co-occurrence of ARGs and Phylogenetic groups in dryland

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and paddy soil. Networks were rendered using Cytoscape v. 3.3.0 based on a significant

(reference)

(ARG)

-

- used depended on the comparison, i.e. effect of

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Spearman correlation (ρ > 0.7, p-value < 0.05) of ARGs, MGEs and bacteria.

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analyses were performed using R 3.1.3

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selected at p < 0.05.

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All

, and the threshold for significance was

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ARGs, MGEs and Antibiotics in Soil and Manure. The numbers of different genes

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associated with ARGs and MGEs and their abundances were considerably higher in the

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composted manure to be applied compared to those in other samples (Fig. 1-2, S1, S2),

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indicating inoculation of ARGs carrying bacteria or DNA into the composted-manure

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applied soil. Furthermore, antibiotics were detected in both the manured dryland and

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paddy soils, but not in the unfertilized soil, and chemically fertilized soil (Fig. 3),

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indicating the long-term use of antibiotics in the production cycle and the potential for

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continued selection for antibiotic resistance in the field.

RESULTS AND DISCUSSION

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The absolute gene copy numbers of ARGs and MGEs in the paddy soil were

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generally one order of magnitude higher per g dry wt than those in the dryland soil for

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the same treatment (Fig. 1, S1). In the dryland soil, long-term application of chemical

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fertilizer or composted manure increased the gene copy number of ARGs and MGEs

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about one order of magnitude over those in the unfertilized soil, whereas in the paddy

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soil, these treatments increased those gene copy numbers only 2-3 times than in the

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unfertilized soil. The gene copy numbers (per g dry wt) of ARGs and MGEs in the

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composted manure were 4 to 5 orders of magnitude higher than in the manure-applied

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dryland and paddy soils (Fig. 1). Similar patterns were observed for primer sets

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targeting the universal 16S rRNA gene indicating a higher bacterial biomass per g dry

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wt of the manured soil. Significant positive Spearman correlations between log 10

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transformed gene copy number of ARGs, and 16S rRNA gene or MGEs in soil were 10


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observed (Fig. 1b), indicating that ARGs abundances correspond to the total microbial

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biomass in soil. For better comparison, ARGs were normalized by the bacterial mass

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(Fig. 2). Due to the low values of 16S rRNA genes in the dryland soil without

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fertilization (Fig. 1), the relative abundance of some ARGs appears to be higher than

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the soil applied with the composted manure (Fig. 2).

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Influence of Fertilization on ARGs Composition in Soil. In general, similarly low

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levels of relative abundances of ARGs and MGEs in the soil was observed regardless

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of the application of either chemical fertilizer or composted manure (Fig. 2). This

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indicates that there is not a major buildup of ARGs in any treatment after 26 years,

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which is documented by the mainly low fold-change of ARGs in the soil applied with

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chemical fertilizer or composted manure compared to the unfertilized soil (Fig. S3).

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Hence, manure from antibiotics-treated animals has not resulted in a strong

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environmental burden of antibiotic resistance genes, but the ARGs-containing refugia

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may still persists capable of growth if selective conditions appear. There are, however,

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some lesser or minor effects of the studied treatments on ARG types and abundances.

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There was a greater difference between ARGs in dryland soil vs. paddy soil than due to

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chemical fertilizer or composted manure applications (Fig. S3, S4a), indicating that the

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effect of different crops (aerobic vs. anaerobic soil) was stronger than the effect of

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different fertilization. Compared to the unfertilized soil, long-term application of

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chemical fertilizer in the dryland soil led to increased abundance of some multidrug, β-

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lactam and tetracycline resistance genes although no antibiotics were applied (Fig. 4a,

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S6a). However, some genes decreased including multidrug sat4, sulfonamide sul3,

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aminoglycoside aphA3_1 and plasmid IncP_oriT gene (Fig. 4a, S6a). On the other hand,

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Compared to the unfertilized soil, chemical fertilization showed mainly depletion of 11



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ARGs in the paddy soil (Fig. 1-4, S3), in which higher pH, organic matter content and

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available P should have favored bacterial growth as shown by the higher microbial

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biomass (Table S1, Fig. 1). Hence chemical fertilization resulted in enrichment of some

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ARGs in the dryland soil, but enhanced dissipation of some ARGs in the paddy soil,

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which might be due to different dynamics of bacteria conferring resistance sequences

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in the dryland vs. the paddy soil. Mineral fertilization (nitrogen) was reported to

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significantly enhance tetA abundance in a grassland soil.

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metagenomics shows that in high-N soils, β-lactamases were depleted as high N levels

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favor particular organisms, leading to shifts in bacterial abundances, which may affect

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resistome composition. 23

12

In contrast, functional

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There was an increase over the non-amended control of some ARGs (Fig. 3a).

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However, some ARGs in the composted manure applied soil were lower compared to

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abundance in the non-manured soil (Fig. 2, S3), showing that manure does not always

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increase ARGs in soil, which is confirmed by the absence of correlation between

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relative abundances of ARGs in composted manure vs. composted manure-applied soil.

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Previous studies have shown that ARGs introduced by manure in dryland soils undergo

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either an increase followed by a decrease or an exponential decrease in the field. 38-41 A

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period of up to a year may be sufficient for some of the gene markers (e.g. sul1, intI1

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and intI2) to approach stable background levels. 12, 41 Whereas, the dynamics and fate

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of ARGs in paddy soil introduced by manure is still unknown.

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We specifically compared some ARGs related to the antibiotics detected in the soil

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(Fig. 3). Of the tetracycline resistance genes, most of them, including tetM, tetO and

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tetT, were detected in both unfertilized soils. Their abundances, however, increased

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after the long-term application of chemical fertilizer or manure, in which manure played

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a larger role than chemical fertilizer (Fig. S3a, S7a). However, gene copy number of 12


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tetL and tetA(P) decreased in both the dryland soil and the paddy soil with chemical

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fertilizer application (Fig. S3a, S7a), which might be due to the negative effect on the

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bacteria carrying these genes. Sulfonamide resistance genes (folP, sul1, sul2 and sul3)

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were essentially unchanged regardless of fertilized or unfertilized paddy soils, but their

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gene copy numbers in fertilized dryland soils were higher than in unfertilized dryland

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soils (Fig. S3a, S7b). The ermB-conferring tylosin resistance most likely originated

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from composted manure applied to the field (Fig. S3a, S7c).

268 269

Influence of Different Cropping-system (aerobic vs anaerobic conditions) on

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ARGs in Soil. Compared to the unfertilized dryland soil, more ARGs with higher

271

abundance were observed in the unfertilized paddy soil (Fig. 1, 2), agreeing with the

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fold-change results showing that a majority of ARGs except for aadE and ampC

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increased in the unfertilized paddy soils (Fig. 4b). Thus, different populations were

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selected in the aerobic and anaerobic conditions and the paddy field with higher

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bacterial populations had a larger reservoir of ARGs (Fig. 1). However, when chemical

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fertilizer was applied, a lower relative abundance of ARGs (Fig. 2) was detected in the

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paddy soil than the dryland soil. When composted manure was applied, higher relative

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abundances of ARGs (Fig. 2) due to higher microbial biomass were detected in the

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paddy soil compared to the dryland soil. However, particular ARGs (such as mexF, oprJ,

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tetPA and acrA_5) increased while others decreased (such as aacC, tetO_2, floR and

281

sat4) (Fig. 4b, S3b), showing that although different cropping system did not affect the

282

overall pattern of ARGs in the soil, different ARGs - likely their host populations -

283

which might have originated from the composted manure or intrinsic soil may undergo

284

different fates due to their different physiologies under aerobic vs. anaerobic conditions.

285 13



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ARG Diversity and Similarity. The primer sets used in this study are more extensive

287

compared to many other studies, permitting a more comprehensive evaluation of ARGs

288

in soils. The Shannon index (diversity) of ARGs and MGEs in the dryland soil was

289

lower than the paddy soil when no fertilizer was applied (Fig. 5), showing that long-

290

term anaerobic conditions increased the biodiversity of the intrinsic antibiotic resistome

291

which corresponded to a higher bacterial diversity in the paddy soil. However,

292

compared to the soil without fertilization, long-term application of chemical fertilizer

293

or composted manure increased the Shannon diversity of ARGs and MGEs in the

294

dryland soil, whereas they did not change the diversity in the paddy soil, confirming

295

the different fate of ARGs in soil introduced by composted manure under aerobic and

296

anaerobic conditions.

297

Six ARGs (acrA_5, fox5, oprJ, sat4, ermF and suld) and two MGEs (IncP_oriT and

298

pNI105map) were detected in both unfertilized soils and irrigation water (Fig. S2c) and

299

their gene copy numbers in paddy soil were higher than in dryland soil (Fig. S1),

300

showing that these genes might be introduced by bacteria in the irrigation water. Thirty-

301

four ARGs present in the irrigation water were not detected in the unfertilized dryland

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soil nor paddy soil (Fig. S2c), showing that the irrigation water may not lead to long-

303

term establishment of ARGs in the soil. Likewise, 141 of the 210 ARGs and MGEs

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present in the composted manure were not detected in any soil (Fig. S2d). However,

305

more than 20 ARGs and MGEs, which were detected in the composted manure, but not

306

present in the unfertilized soil, were detected in both the composted manure applied

307

dryland and paddy soils (Fig. S2d), indicating that these genes might have originated

308

from bacteria in the manure. Among all soil samples, 12 ARGs were exclusively found

309

in the chemical fertilized dryland soil (Fig. S2e), inferring that long-term chemical

310

fertilizer application selected for certain ARG carrying aerobic bacteria. Four ARGs 14


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blaFOX, acrA_5, mexF, and oprJ - and one MGE - pNI105map - were shared in all soil

312

samples, manure, and water (Fig. 2, S2), indicating that they might be part of a global

313

natural resistome, agreeing with our report on natural ARGs in Antarctic soil. 29

314 315

Co-occurrence of ARGs, MGEs and Phylogenetic Groups. In both the dryland soil

316

and the paddy soil, pNI105map - a small plasmid MGE 26 – co-occurred with several

317

ARGs (Fig. S5), inferring that horizontal gene transfer might be responsible. The genera

318

with the most co-occurrence of ARGs and MGEs are Bradyrhizobium and

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Acidobacteria Gp1 followed by Gemmatimonas in the dryland soil (Fig. S5a).

320

Bradyrhizobium is reported to be resistant to multiple antibiotics. 42 The abundance of

321

Gemmatimonas increased in the soil amendment repeatedly with sulfadiazine

322

containing manure. 43 In the paddy soil, Acidobacteria Gp2 clade was the taxa that most

323

co-occurred with ARGs (Fig. S5b). Acidobacteria is one of the most widespread and

324

abundant phyla in the soil, 44 but a large number of genes in the few strains studied are

325

associated with MGEs-encoding transposases, insertion sequence elements, and phage

326

integrases. 45

327 328

Influence of Soil Characteristics, Antibiotics and Bacterial Diversity on ARGs. Soil

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physiochemical properties were measured to investigate trends among ARGs

330

abundance, bacterial diversity and edaphic factors under different fertilization and

331

cropping system (Table S1). Bacterial Shannon index is the most significant attribute

332

that correlates with the relative abundance of ARGs and MGEs in the soil (Fig. S8,

333

Table S2), which is consistent with a more diverse bacterial structure contributing to

334

more diverse ARGs.

335

relative abundance of ARGs in the soil, perhaps due to the persistence of Tetracycline

29

Antibiotics (Carbadox and Tetracycline) correlate with the

15



Page 16 of 30

336

46-48

and the genotoxicity and specificity of Carbadox. 49 A significant correlation was

337

observed between the concentration of arsenic, which is often an additive in swine feed,

338

and relative abundance of ARGs in soils. Soil pH was significantly correlated with

339

ARGs, which is expected as soil pH is a well-known factor affecting microbial

340

community structure, including at this location,

341

bioavailability and speciation of some antibiotics. 51,52

50

and may also influence

342 343

Environmental Implications. Long-term application of composted swine manure

344

containing antibiotics did not build up a major ARGs burden in soil regardless of

345

aerobic (dryland) or anaerobic (paddy rice) conditions. The soil is both a natural

346

reservoir as well as a sink for ARGs; the later likely reduces the risk of ARGs introduced

347

by manure, sludge or wastewater. However, other soil management practices can also

348

affect the soil resistome. The occurrence and fate of ARGs (and their host microbes)

349

are influenced by soil oxygen status, nutrient and organic carbon additions, cropping

350

systems, and other factors that drive microbial community structure. Therefore, the

351

origin, dynamics, and transport of ARGs in fields with different agricultural practices

352

and soil types is useful for understanding the dynamics of intrinsic and introduced

353

ARGs in soil and therefore inform risk mitigation strategies.

354 355



356

Supporting Information

357

Supporting Information is available free of charge via the Internet at http://pubs.acs.org.

358

Gene copy number of ARGs, ARGs share, relationship between gene copy numbers of

359

ARGs and 16S or MGEs, fold change of ARGs in soil with different fertilization and

360

cropping system, redundancy analysis of ARGs and bacteria, co-occurrence network of

ASSOCIATED CONTENT

16


Page 17 of 30

361


ARGs and bacteria, physicochemical properties of composted manure and soil.

362 363



364

Corresponding Authors

365

*E-mail

366

[email protected].

367

Notes

368

The authors declare no competing financial interest.

AUTHOR INFORMATION

address:

Fang

Wang,

[email protected];

James

M.

Tiedje,

369 370



371

The research was funded by the National Natural Science Foundation of China

372

(21677149), the Outstanding Youth Fund of Natural Science Foundation of Jiangsu,

373

China (BK20150050), the Innovative Project of the Chinese Academy of Sciences

374

(ISSASIP1616),

375

(2016YFD0800204), the Key Program of Frontier Sciences of the Chinese Academy of

376

Sciences (QYZDJ-SSW-DQC035), and the Center for Health Impacts of Agriculture

377

(CHIA) of Michigan State University.

ACKNOWLEDGMENTS

the

National

Key

Basic

Research

Program

of

China

378 379



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25



Page 26 of 30

1.0E+12

a

ARG

Gene copy number (per g dw or ml)

1.0E+11

d

16S

MGE

1.0E+10 1.0E+09 1.0E+08 1.0E+07

b

1.0E+05

b

c

b

b

b b

1.0E+06

c

b

b b

c

bc

b

d

e

b

b

b

a

b

1.0E+04 1.0E+03

545

a a DNF

DCF

DMF

WNF WCF Treatments

WMF

Manure

Water

b

546 547

Figure 1 Gene copy numbers (per g dry weight or ml) of antibiotic resistance genes

548

(ARGs), mobile genetic elements (MGEs) and 16SrRNA in the composted manure,

549

irrigation water and soil (n = 3), one-way analysis of variance was conducted among

550

different treatments for ARG, MGE and 16SrRNA, respectively (a) and the relationship

551

between log-transformed (base 10) gene copy numbers of ARGs and 16S or MGEs in

552

soil (b). DNF, dryland soil with no fertilizer; DCF, dryland soil with chemical fertilizer;

553

DMF, dryland soil with composted manure; WNF, paddy soil with no fertilizer; WCF,

554

paddy soil with chemical fertilizer; WMF, paddy soil with composted manure.

26


Page 27 of 30


MGE Other Vancomycin

Tetracycline Sulfonamide

MLSB

MDR

tnpA_4 tnpA_3 tnpA_2 tnpA_1 repA IncP_oriT IncN_rep orf39-IS26 ISPps ISEfm1 IS1111 nimE nisB vanXD vanWG vanTG vanTC vanSB vanHB vanC vanB tetX tetW tetT tetR tetPB tetO_2 tetO_1 tetM_3 tetM_2 tetM_1 tetL tetG_2 tetG_1 tetA/B tetA(P) tetA tet34 tet(32) sul3 sul2_2 sul2_1 sul1 folP_3 folP_2 folP_1 vgaB_2 vgaB_1 vatE vatC pncA pikR2 oleC msrA_2 msrA_1 mphA_2 mphA_1 matA/mel lnuB_2 lnuB_1 ermT ermF yceL/mdtH sat4 qacE△1_3 qacE△1_2 qacE△1_1 oprJ oprD mtrE mtrC mexF mepA marR emrD emrB/qacA acrR acrA_3 acrA_2 acrA_1 floR cmlA ceoA pbp cphA_2 cphA_1 blaSFO blaPER blaIMP blaFOX blaCTX-M_2 blaCTX-M_1 blaCMY blaACT ampC aphA3_2 aphA3_1 aadE aadA_6 aadA_5 aadA_4 aadA_3 aadA_2 aadA_1 aacC4 aac3-VI aac(6')-II

RA (%) 5x10-4 1x10-3 5x10-3 1x10-2 5x10-2 1x10-1

Chloramphenicol

Beta Lactam

Aminoglycoside

5x10-1 1x100 5x100 1x101 5x101

DNF

DCF

DMF

WNF

WCF

WMF

Manure

Water

555 556

Figure 2

557

and mobile genetic elements to 16S rRNA genes in the composted manure, irrigation

558

water and soil with different fertilizer treatments and different cropping system. The

559

resistance genes detected in composted manure or water, but not in the soil were not

560

shown. Sample code is the same as in Figure 1.

Heatmap shows relative abundance (RA, %) of antibiotic resistance genes

561

27



562 563 564

Figure 3 Antibiotics detected in the dryland soil and paddy soil 1 year after application of composted manure.

28


Page 28 of 30

Page 29 of 30


a

Amphenicol

Beta Lactam

Aminoglycoside

MGE

MDR MLSB

-20.88

Fluoroquinolone

0

18.34

Log2 fold of change unknown Sulfonamide Tetracycline

Vancomycin

Observed in WMF vs WNF Observed in WCF vs WNF Observed in DMF vs DNF Observed in DCF vs DNF

MDR

b Aminoglycoside

Beta Lactam

MLSB

unknown

Amphenicol Vancomycin

-18.09

MGE

0

17.73

Log2 fold of change Observed in WCF vs DCF Observed in WNF vs DNF Sulfonamide

565 566 567 568 569 570

Fluoroquinolone

Tetracycline

Observed in WMF vs DMF

Figure 4 Differentially observed antibiotic resistance genes in the soil with different fertilizer compared to the control (no fertilizer) (a) and paddy soil compared to the dryland soil (b) based on Log2 transformed fold of change. Red means ARGs are higher, blue means ARGs are lower with the color intensity indicating extent of change. Sample code is the same as in Figure 1.

29



571 572

Figure 5 Shannon index of relative abundance of antibiotic resistance genes and mobile

573

genetic elements (a) and microbial community structure (b) in the dryland soil and

574

paddy soil with no fertilizer (NF), chemical fertilizer (CF) and composted manure (MF).

30


Page 30 of 30

Long-Term Effect of Different Fertilization and Cropping Systems on

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