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Soil Biology and Biochemistry

journal homepage: www.elsevier.com/locate/soilbio

Application of biosolids drives the diversity of antibiotic resistance genes insoil and lettuce at harvest

Lu Yanga,b, Wuxing Liua, Dong Zhub,c, Jinyu Houa,b, Tingting Mad, Longhua Wua,∗,Yongguan Zhuc, Peter Christiea

a Key Laboratory of Soil Environment and Pollution Remediation, Institute of Soil Science, Chinese Academy of Sciences, 71 Beijing Road, Nanjing 210008, ChinabUniversity of the Chinese Academy of Sciences, 19A Yuquan Road, Beijing 100049, Chinac Key Laboratory of Urban Environment and Health, Institute of Urban Environment, Chinese Academy of Sciences, 1799 Jimei Road, Xiamen 361021, Chinad Institute of Hanjiang, Hubei University of Arts and Science, 296 Longzhong Road, Xiangyang 441053, China

A R T I C L E I N F O

Keywords:AntibioticsAntibiotic resistance genesARGsBiosolid applicationLettuceSoil

A B S T R A C T

High-throughput quantitative polymerase chain reaction (PCR) of antibiotic resistance genes (ARGs) was used toprofile the composition and diversity of ARGs in biosolid-amended soil and in lettuce roots and shoots. Biosolidapplication significantly increased the ARGs in soil and influenced the soil antibiotics resistome mainly throughexogenous introduction. Four dominant ARGs occurred in sterilized and water-washed leaves, namely catB8,php, vanB and str, which accumulated to> 50% of total abundance. Nine subclasses of ARGs were determined inboth sterilized and water-washed leaves, and they were shared ARGs among soil, roots and shoots. This suggeststhat the ARGs in ready-to-eat lettuce were mainly located in endophytes and the internal route may contributemore to the ARGs in routinely consumed raw lettuce. Moreover, a previously unsuspected shift in multridrugARGs to root and shoot compartments due to biosolid application was discovered. These results reveal thatbiosolid application drives the evolution and dissemination of ARGs in the soil-plant system.

1. Introduction

Land application to agricultural fields is a common approach for therecycling of plant nutrients in biosolids. The current regulations inChina give priority to the control of inorganic and organic pollutants inbiosolids for agricultural use (Ministry of Environmental Protection ofthe People's Republic of China, 1984; Standardization Administration ofthe People's Republic of China, 2009). According to the Disposal ofSludge from Municipal Wastewater Treatment Plants - Control Standards forAgricultural Use (CJ/T 309–2009), Class A biosolids are licensed for theamendment of agricultural soils for vegetable and cereal production(Standardization Administration of the People's Republic of China,2009). Some recent investigations show decreasing concentrations ofpotentially toxic metals and organic pollutants in Chinese biosolids(Meng et al., 2016; Yang et al., 2015; Cheng et al., 2014). However,domestic biosolids are hotspots of antibiotic resistance genes (ARGs)(Rodriguez-Mozaz et al., 2015; Su et al., 2015; Michael et al., 2013;Zhang and Zhang, 2011). Thus, increasing application of biosolids maybe carried out legally with no regard for the risk of disseminating ARGsinto agricultural products.

The occurrence of antibiotic resistance genes is natural (Martínez,

2008). However, human activities such as biosolid application accel-erate the evolution and dissemination of resistance. On one hand,exogenous ARGs might remain in soil microorganisms (Rahube et al.,2016; Heuer et al., 2008) due to their co-resistance to other pollutantsconferring a survival advantage to their host (Stepanauskas et al.,2005). For example, an efflux pump may work for resistance to bothantibiotics and potentially toxic metals (Martínez et al., 2009; Baker-Austin et al., 2006). On the other hand, indigenous ARGs may be sti-mulated by nutrients and other chemical compounds in addition toantibiotics (Chen et al., 2017a; Hu et al., 2016, 2017).

Application of biosolids to agricultural land may raise human ex-posure to ARGs. Consumption of raw leaf vegetables may therefore be aroute of increasing direct human exposure to ARGs (Wang et al., 2015;Rahube et al., 2013). Uncooked vegetables such as lettuce are popularin salads. ARGs can spread with endophytes or by adhering to plantsurfaces (Chen et al., 2017b; Yang et al., 2016). The genes sul2, ermE,strA and tetT have been detected in vegetables grown on land to whichbiosolids have been applied (Lau et al., 2017). The ARGs tetC, tetD,qacH, aadD and emrB have been reported to show higher risks of ad-hesion to edible plants with organic fertilizer amendment (Lau et al.,2017; Rahube et al., 2016). Zhu et al. (2017) detected higher

https://doi.org/10.1016/j.soilbio.2018.04.017Received 5 January 2018; Received in revised form 13 April 2018; Accepted 16 April 2018

∗ Corresponding author.E-mail address: lhwu@issas.ac.cn (L. Wu).

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abundance of ARGs in organic than in conventionally produced lettuce.However, the influence of biosolid application on the evolution anddistribution of ARGs in arable crops remains to be investigated.

The resistome is the aggregation of ARGs in an ecosystem and it canprovide an early warning of emerging and multiple resistances (D'Costaet al., 2006). Previous studies have focused on the risks of biosolidapplication in regard to changes in the abundance of ARGs but have notaddressed the evolution of the resistome (Lau et al., 2017). Currentantibiotic resistance problems always involve multiple resistance. Fur-ther research is therefore required to study the variation in the re-sistome.

The dissemination of ARGs depends mainly on the growth of mi-crobes. The ARG profile and bacterial community might co-evolve witha changing territorial environment (Wu et al., 2017; Laskaris et al.,2010). However, various bacterial phyla are found to be related to theevolution of ARGs (Pu et al., 2018; Xiong et al., 2018; Chen et al., 2016;Huerta et al., 2013). Indeed, the horizontal gene transfer of plasmidsmay contribute to the dissemination of ARGs and this suggests thatthere will be higher risks of the prevalence of ARGs following biosolidapplication.

The present study was designed to profile the evolution and dis-tribution of ARGs driven by biosolid application. Targeting of 295primer sets was involved to comprehensively represent the antibioticresistome in soil, roots, shoots and ready-to-eat lettuce. Illumina se-quencing of 16 S rRNA gene-base communities was conducted. Thespecific objectives were to investigate the impacts of biosolid applica-tion on the abundance and structures of ARGs in soil and lettuce atharvest, to study the distribution of ARGs in lettuce, and to deduce themain patterns of the evolution and distribution of ARGs following ap-plication of biosolids.

2. Materials and methods

2.1. Pot experiment and sampling

The rooting zone (top 15 cm) of a Typic Hapli-Stagnic Anthrosolsprofile was sampled at Suzhou city (31.45° N, 120.43° E), Jiangsuprovince, east China. After air-drying and sieving through a 4-mmnylon mesh, the soil was placed in plastic pots. Biosolid samples werecollected from a domestic wastewater treatment plant in Suzhou city.The biosolid samples were composted in the shade until they reachedthe standard moisture content (CJ/T 309–2009,< 60%). They werestirred every other day to minimize their heterogeneity. Physico-che-mical properties of the test soil and biosolids were determined beforemixing and are shown in Table 1. Seeds of the test lettuce (Lactucasativa L. var. ramosa Hort.) were purchased from Jinshengda Seed Co.Ltd., Nanjing, east China.

The pot experiment was conducted in a glasshouse at the Institute ofSoil Science, Chinese Academy of Sciences, Nanjing, Jiangsu province.The temperature was controlled at 30 °C throughout the day with illu-mination of 10000 lux for 16 h per day. Each pot contained 2.0 kg ofsieved soil (oven dry basis). The application rates of the biosolids as apercentage of the soil weight (DM basis) were 0 (control), 0.3, 1.3 and3%. The moisture content of the biosolids was 60%. The biosolids andsoil were mixed 40 days before sowing of the seeds. Three lettuce plantswere grown in each pot and there were three replicate pots of eachtreatment. Deionized water was added daily to maintain the soil at 70%of water holding capacity (WHC). The lettuce was harvested after 63days of growth.

Fresh soil samples were collected at the lettuce harvest and pre-pared immediately by flash freezing using liquid nitrogen. They werethen freeze-dried in a FreeZone 4.5 L (Labconco Company, Kansas City,MO). Each harvested whole lettuce was washed with tap water and thensterilized Milli-Q water (18.2MΩ cm, 25 °C). The shoot was dividedinto two equal parts, one of which was surface sterilized together withthe roots and the other was flash frozen and stored freeze-dried. Surface

sterilization was done according to Zimmerman and Vitousek (2012).Soil samples were stored at −80 °C prior to determination of anti-

biotics and DNA extraction. Other portions were stored at room tem-perature for determination of soil physicochemical properties and totalconcentrations of potentially toxic metals.

2.2. Determination of soil physicochemical properties

Soil pH, cation exchange capacity (CEC), organic matter content(OM), available NPK, and total NPK were determined using the stan-dard methods described by Sparks et al. (1996).

2.3. Determination of potentially toxic metals in soil and plants

Soil and lettuce samples were digested using a modification of thehigh-pressure sealed digestion method (Zhou et al., 2016). The testsamples were ground and sieved through a 0.15-mm nylon mesh. Theunsterilized shoots were used for trace metal analysis. Subsamples ofsoil (0.2000 g) or shoots (0.5000 g) were digested in 8-mL acid mixture(soil, HNO3: HF: H2O2, 5:2:1 (v/v/v); shoots, HNO3:H2O2, 6:2 (v/v) in ahigh-pressure sealed reaction vessel. The digests were diluted to 10mLwith Milli-Q water (18.2 MΩ cm, 25 °C) and filtered through a 0.45-μmfilter. The metal concentrations were determined by ICP-MS (7700x,Agilent Technologies, Santa Clara, CA). Certified reference materialsGBW07429 and GBW10020 (National Geochemical Standard Materials,China) were used for QA/QC of soil and plant digestion, respectively.

2.4. Determination of antibiotics in soil

Tetracycline (TC), oxytetracycline (OTC), chlortetracycline (CTC),doxycycline (DOC), norfloxacin (NFC), ofloxacin (OFC), ciprofloxacin(CFC), enrofloxacin (EFC), roxithromycin (RTM), sulfadiazine (SDZ),sulfadimethoxine (SDM), sulfaclozine (SCZ), sulfaquinoxaline (SQX),sulfameter (SM), sulfamonomethoxine (SMM), sulfamethoxazole (SMX)and sulfamethazine (SMZ) were determined by methods describedpreviously (Huang et al., 2013).

2.5. Soil DNA extraction

Soil total DNA was extracted from 0.5-g samples and total shoot

Table 1Antibiotics in the selected biosolids and soil (μg kg−1). The biosolid sampleswere collected before application and soil samples were collected at the vege-table harvest.

Antibiotics Biosolids Control 0.3%SS 1.3%SS 3%SS

TetracyclinesTetracycline (TC) 644 2.19 2.48 2.49 8.66Oxytetracycline (OTC) 1714 2.22 4.88 8.81 15.05Chlortetracycline (CTC) 520 1.98 2.43 2.06 7.07Doxycycline (DOC) 64.8 1.20 1.95 3.09 2.31FluoroquinolonesNorfloxacin (NFC) 3892 13.15 28.68 47.53 99.03Ofloxacin (OFC) 2225 4.36 11.24 22.93 46.03Ciprofloxacin (CFC) 1399 8.28 9.70 13.95 21.98Enrofloxacin (EFC) 49.6 3.09 3.26 3.28 4.39MacrolidesRoxithromycin (RTM) 56.3 0.21 0.19 0.31 0.34SulfonamidesSulfadiazine (SDZ) 6.55 – – – –Sulfamethoxazole (SMX) 4.97 – – – –Sulfamethazine (SMZ) 5.41 – – – –Sulfamonomethoxine (SMM) 1.43 – – – –Sulfaquinoxaline (SQX) 5.04 – – – –Sulfadimethoxine (SDM) 3.44 – – – –Sulfaclozine (SCZ) 6.44 – – – –Sulfameter (SM) 3.68 – – – –SUM 10602 36.7 64.8 104 205

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DNA from 0.2-g samples using FastDNA SPIN Kits (MP Biomedicals,Santa Ana, CA). The quality and concentration of DNA were determinedusing a NanoDrop ND-1000 spectrophotometer (Thermo Fisher,Waltham, MA) and QuantiFluor dsDNA system (Promega, Madison,WI). The DNA samples were stored at −20 °C until use.

2.6. High-throughput quantitative PCR

A Wafergen SmartChip Real-time PCR system (Wafergen, Fremont,CA) was used to perform the high-throughput quantitative PCR of theARGs. After a hot start at 95 °C for 10min, 40 cycles of repeated am-plification were carried out with denaturation at 95 °C for 30 s andannealing at 60 °C for 30 s according to previous studies (Wang et al.,2014). There were 296 primer sets involved targeting 213 antibioticresistance genes (ARGs), 10 mobile genetic elements (MGEs) and 1 16 SrRNA gene (Table S1). The detection limit was set to a threshold cycle(Ct) of 31. Three technical replicates were made for each sample. Thesamples were positive for quantification when Ct values of at least tworeplicates were above the detection limit. The gene copy number wascalculated by 10(31−Ct)/(10/3), and the relative abundance of ARGs wascalculated by dividing the targeted ARG copy number by that of 16srRNA. ARG profiles were categorized into eight defined classes of an-tibiotic resistance (Chen et al., 2016).

2.7. Illumina sequencing and analysis of 16s rRNA gene

Soil bacterial 16s rRNA (V4-V5) was amplified using primers 515Fand 907R. The PCR reactions were performed using a TransGen AP221-02 reaction system (Destin, FL) with TransStart Fastpfu DNAPolymerase, 20 μL with an ABI GeneAmp 9700 (Thermo Fisher,Waltham, MA). The PCR products were quantified using a QuantiFluordsDNA system (Promega, Madison, WI). The amplification productswere then pooled and submitted for sequencing using an Illumina Miseqplatform (Majorbio, Shanghai, China). A Trimmomatic trimmer wasused to trim amplicon sequencing data (Bolger et al., 2014). Fast LengthAdjustment of SHort reads (FLASh) 1.2.11 was used to assemble thereads (Magoč and Salzberg, 2011) and 30832-44283 high quality se-quences were acquired from all samples. The generated high-qualitysequences were further analyzed by QIIME with the default parameter(Caporaso et al., 2010). The open-reference operational taxonomicunits (OTUs) were clustered by UCLUST at the 97% similarity level(Edger, 2010). The taxonomic classification was performed using RDPclassifier which was trained on the Greengenes version 13.5 16 S rRNAdatabase (McDonald et al., 2012; Wang et al., 2007).

2.8. Statistical analysis

Mean values and standard deviations were calculated usingMicrosoft Excel 2017. The concentrations of antibiotics were normal-ized to acquire Z-scores using Origin Pro 2017. The statistical analysiswas performed using the SPSS version 20.0 software package. Thestatistical results were considered significant at the 5% level. Figureswere generated with Origin Pro 2017. Gephi (0.9.1) was used to vi-sualize the co-occurrence of ARGs in the biologic flow. R with thepackage “pheatmap” was used to analyze the microbial communitystructure in soil and in biosolids. LEfSe (linear discriminant analysiseffect size) was performed online at http://huttenhower.sph.harvard.edu/galaxy (Segata et al., 2011). The alpha value for a factorialKruskal-Wallis test and a pairwise Wilcoxon test were set at 0.05. Athreshold of 2.0 of the logarithmic LDA score was assigned for dis-criminative features.

3. Results

3.1. Effects of biosolid application on changes in soil fertility and pH

Biosolids are a potentially useful source of plant nutrients for leafyvegetables because of their relatively high nitrogen (N), phosphorus (P)and organic matter (OM) contents. As shown in Table S2, soil total Nand P contents increased significantly at 3% application rate(p < 0.05). Soil available N, available P and OM also increased sig-nificantly at both 1.3 and 3% application rates (p < 0.05). Also, theratio of available N increased to 18.1, 24.6 and 23.0% at 0.3, 1.3 and3% biosolid application rates, respectively, and the corresponding va-lues of available P were 7.4, 10.0 and 11.4%, respectively. Soil pHdeclined significantly with biosolid application but the other soilproperties tested did not change significantly at the lettuce harvest.

3.2. Effects of biosolid application on trace metal concentrations in soil andlettuce

Table S3 shows that the trace metal concentrations in soil and let-tuce were affected by increasing biosolid application rate. Althoughzinc and copper accumulated significantly in the soil at 1.3 and 3%biosolid application rates (p < 0.05), they still met the second levellimit of the Chinese Soil Environmental Quality Standard. Moreover,the controlled toxic elements in lettuce, namely Pb, Cd and As, all metthe national food safety standards.

3.3. Residual antibiotics in the soil

A total of nine antibiotics were detected in the test samples(Table 1). All subtypes of the sulfonamides were below the detectionlimits. Residues of NFC, OFC and OTC showed a dose-dependent re-lationship with biosolid application rate. Substantial accumulation ofTC, CTC, CFC, EFC and RTM occurred only at the high biosolid appli-cation rate. As shown in Table S3, the residual rate of fluoroquinoloneswas higher than that of tetracyclines or macrolides. The sum of the totalconcentrations of selected antibiotics (104 and 205 μg kg−1 at 1.3 and3% application rates, respectively) exceeded the ecotoxicity effecttrigger value of 100 μg kg−1 (AHI, 1997).

3.4. ARGs in biosolids and soil

Biosolid application changed the abundance and diversity of ARGs.A total of 150 ARGs and 7 MEGs were detected across all the biosolidand soil samples. The abundance of ARGs and MEGs increased withbiosolid application and reached statistical significance (p < 0.05) atthe 3% application rate (Fig. 1). In the control without biosolidamendment the ARG classes aminoglycosides and beta-lactams weredominant in the soil. The percentages of the ARG classes macrolide-lincosamide-streptogramin B (MLBS), sulfonamides, tetracyclines andvancomycin showed an upward trend with biosolid application.

The up-regulated ARGs and MGEs (p < 0.05) with biosolids ap-plication were selected and compared with their relative abundance inthe biosolids (Fig. 2). There were 19 subtypes whose abundancewas> 0.01. There were five ARGs whose abundances were even>0.10 under the 3% biosolids treatment, namely pikR2, mecA, vanB, aac(6′)-II and tetL. However, the corresponding abundances in the testbiosolids were 0.04, 0.07, 0.02, 0.02 and 0.02, respectively. Most ofthese increased with biosolid application in the sequence control<0.3% < 1.3%<3% biosolids in the soil. Furthermore, some of thesewere rarely detected in the control soil and increased significantly withbiosolid application, for example aac (6’)-II, vanRD, mphA, blaPSE andermB. There remained some soil native ARGs such as aadA-1 whichclearly increased at the 3% biosolid application rate.

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3.5. Characterization of ARGs in lettuce and their transmission pathways

The top 20 ARGs detected on surface-sterilized leaves and sterilizedMilli-Q water-washed leaves are shown in Fig. 3 and ordered from thehighest to the lowest abundance. They comprised>70% of the totalabundance and belonged to seven classes of ARGs, namely aminogly-cosides, beta-lactams, MLSB, multidrug sources, tetracyclines, vanco-mycin and others. catB8, php, vanB and str were the same dominant fourARGs in both treatments. aadA1, aadA2, rarD and acrB were also foundin both surface-sterilized and sterilized Milli-Q water-washed treat-ments but their abundances in sterilized water and washed leaves were1.0–13 times higher. In the surface-sterilized treatment the numbers ofsubtypes were similar across the different classes of ARGs (Table S4).However, more aminoglycosides and beta-lactams were found in thetop 20 ARGs of the sterilized Milli-Q water washing treatment, makingup 50% of the total.

Shared ARGs are shown in Fig. 4 to detail the intrinsic pathway oftransmission of ARGs. A total of 140 ARGs and 9 MGEs were detected inall test samples. Specifically, 50 ARGs were detected in soil, roots andshoots; 27 ARGs were shared between soil and roots; 5 ARGs wereshared between roots and shoots; 5 ARGs were shared between soil androots; and 32, 15 and 4 ARGs were unique to soil, roots and shoots,respectively. Turning to the MGEs, tnpA-04 and IS613 were found insoil, root and shoot samples; TP614, IntI-1 and tnpA-03 co-occurred inboth soil and roots; tnpA-05 and tnpA-07 appeared in soil only; andcIntI-1 and tnpA-02 were specialists in roots and shoots, respectively.

Comparing the top 20 ARGs with co-occurrence in Fig. 4, most ofthe top 20 ARGs were shared in all three compartments (soil, roots andshoots). Interestingly, vgaA and nimE were unique to shoots but tetK andacrA were found in both roots and shoots as part of the top 20 ARGs ofsurface-sterilized leaves. In contrast, aacC and mtrC were unique to soiland belonged to the top 20 ARGs of the sterilized Milli-Q water-washedleaves. The ARGs in sterilized leaves were traced to the roots but soildust will have contributed to the ARGs in water-washed leaves. Overall,this result suggests a risk from the routine consumption of raw lettuce.

The shared 50 ARGs were used to profile the shifts in the resistome.As shown in Fig. 5, the co-occurring ARGs were clustered together inthe control. However, they divided into two groups with biosolid ap-plication. This calculation shows a decreasing contribution of the soil-enriched ARGs and a relative up-regulated expression of ARGs in theroots. In particular, the multidrug-resistant genes showed enhancedtransmission to lettuce endophytes with biosolid application.

3.6. Bacterial communities in soil and lettuce

The bacterial abundance in biosolids was significantly higher thanin the soil (p < 0.05) (Fig. S1). The number of bacteria in soil increasedwith increasing biosolid application rate, namely 4.68×107,4.91×107, 7.13×107 and 9.46×107, respectively, from the controlup to 3% biosolids amendment. The highest application rate (3%) in-duced a significant increase in soil microbial numbers (p < 0.05).

Biosolid application changed the soil bacterial communities (Fig.S1). To understand the changes in the soil bacterial community fol-lowing biosolid application the OTUs>1% at family level were se-lected and are shown in Fig. S2. The taxa which were present in highrelative abundance (RA) in the biosolids and yet low in the control soilwere considered to be biosolid-borne. The taxa with low relativeabundance in the biosolids and high in the control soil were regarded asindigenous. Three categories of OTUs were recognized significantly atfamily level based on hierarchical cluster analysis. They were thendescribed top-to-bottom as i) four biosolid-borne taxa that increasedwith biosolid application, likely related to ARGs introduced into thesoil; ii) twenty indigenous taxa that decreased in the soil, indicatingthat they were inhibited by biosolid application; and iii) twelve soilindigenous taxa that were stimulated by biosolid application.

Shifts in the soil microbiota were further assessed by performing t-tests (p < 0.05) and elucidating the contributions of different phyla(Fig. 6). Application of biosolids increased the abundance of most fa-milies in the Actinobacteria and Bacteroidetes. The abundance of fa-milies in the Acidobacteria, Chloroflexi and Gemmatimonadetes de-creased significantly with biosolid application. Although some familiesin the Proteobacteria decreased with biosolid application, the cumula-tive abundance of Proteobacteria still increased (Fig. S1). Xanthomo-nadaceae (belonging to the Gammaproteobacteria) was the family withthe most significantly increasing relative abundance and the incrementwas up to 11.2%. The four identified endogenous taxa were categorizedinto three different phyla. Although their relative abundance increasedwith biosolid application, the increment was always< 1%, too little tofully explain the amount of increasing ARGs. This suggests that theARGs had undergone horizontal transfer.

The distinction between bacterial communities in the soil, roots andshoots is visualized in Fig. S3. The species had higher diversity in soilthan in the plant parts. Actinobacteria were enriched significantly inthe shoots. The relative abundances of Planctomycetes and Acid-obacteria were high in the soil but these two phyla were scarce in the

Fig. 1. Changes in ARGs and MGEs with different biosolid application rates.

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Fig. 2. Significantly changed ARGs (p < 0.05) in soils with biosolid application and their relative abundance in selected biosolids.

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roots and shoots. Although the taxa enriched in the roots were differentfrom those in the soil, most belonged to the Bacteroidetes and Proteo-bacteria.

4. Discussion

4.1. Impact of biosolid application on soil ARGs

Biosolids supplied abundant nitrogen, phosphorus and organicmatter and might therefore be a potential fertilizer for leafy vegetables.However, they might be considered unsuitable when antibiotics andARGs are taken into consideration. Although the concentrations of in-dividual antibiotics were low, the sum of the concentrations often ex-ceeded the ecotoxicity effect trigger value. Furthermore, antibiotics canstimulate ARGs in soil at low concentrations (Andersson and Hughes,2014) and different antibiotics might act synergistically. Our resultsshow that the total concentrations of antibiotics reached 104 and205 μg kg−1, respectively, in the soil with 1.3 and 3% biosolidsamendment and they may serve to sustain the soil resistance level(Jechalke et al., 2013; Heuer et al., 2009). The dominant ARGs in thebiosolids were also present in high abundance in the test soil and in-creased with increasing biosolid application rate (Fig. 2). Most, forexample aac (6’)-II, vanRD, mphA, blaPSE and ermB, may be consideredto be external genes because they were rarely detected in the controlsoil. The abundance of these ARGs increased to>0.01 following bio-solid application. Stimulated native ARGs such as aadA-1 were in arelative minority. Even the most abundant ARG in control soil, aadA1,was not detected at 1.3 and 3% application rates. This suggests thatexternal loading may have been the main source in the biosolid-amended soil.

4.2. Impact of biosolid application on transmission of ARGs

The enriched ARGs in the soil were able to spread to the lettuceplants (Figs. 3 and 4). Washing lettuce with sterilized Milli-Q water wasintended to simulate the process of washing vegetables in order todistinguish the ARGs derived from the biosolids from those that mightbe introduced through tap water. Surface sterilization and sterilizedwater washing treatments shared the same dominant ARGs, namelycatB8, php, vanB, str and aadA1. This indicates that ARGs in dietary rawleaf vegetables might be chiefly endogenous. Clearly, washing with tapwater cannot remove this portion of ARGs, but the ARGs differed in thesterilized and unsterilized treatments. The 20 dominant ARGs in ster-ilized water-washed leaves belonged mainly to the classes aminogly-cosides and beta-lactamases which comprised>50%. There were moremultidrug, tetracycline and other classes of ARGs in the sterilizedtreatment. This indicates that aminoglycoside and beta-lactamase ARGsmay be more likely to be spread through contamination by soil parti-cles. These differences in the dissemination of different classes of ARGshave been explored to a limited extent in previous studies (Manaia,2017).

4.3. Horizontal transfer of ARGs with biosolid application

As shown in Fig. S2, the bacterial taxa that increased significantlywith increasing biosolid application rate were the indigenous micro-organisms in general. However, the biosolid-borne ARGs were found tobe responsible for the increasing ARG levels in biosolid amended soil.Furthermore, the rate of increase of ARGs did not equal that of the soilmicroorganisms. The rates of increase (F) were calculated as follows:

Fig. 3. Top 20 ARG subtype abundance in (A) surface sterilized leaves and (B) sterilized water-washed leaves.

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=×

× × + ×

R NR N rate R N

F .T T

ss ss 0 0

where, RT, R0 and RSS are the abundances of ARGs or OTUs in treatmentgroups, control group and test biosolids, respectively; NT, N0 and NSS

are the 16rRNA gene copies in treatment groups soils, control soil andtest biosolids; and rate denotes the biosolid application rate. Taking theresults at 3% application rate as an example, pikR2, mecA, aac, vanB andtetL were determined to increase by 160, 56, 106, 183 and 145 times,respectively. The Actinobacteria and Proteobacteria increased by only

2.54 and 2.94 times, respectively. The largest increasing bacterial fa-mily, Xanthomonadaceae, increased only 2.02 times. Clearly, the in-crease in ARGs was not due to a few species. This suggests that theindigenous microorganisms acquired exogenous ARGs with biosolidapplication (Chen et al., 2017c). In other words, HGT may play aleading role in driving the evolution of ARGs in soil with biosolid ap-plication.

The identification of shared ARGs and MGEs provides further evi-dence for horizontal gene transfer. For example, cIntl-1 was unique to

Fig. 4. The co-occurrence patterns among ARG subtypes in soil, roots and shoots.

Fig. 5. Ternary plots depicting ARG distribution in soil, roots and shoots. Each point represents one ARG subtype and its position reflects the abundance with respectto each compartment.

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the roots and Intl-1 was shared by soil and roots. Moreover, sul1 wasunique to soil, sul2 was unique to the roots, and sulA_folP was shared bysoil and roots. The class 1 integron has been found to be significantlyassociated with sulfamethoxazole resistance genes (Aydin et al., 2015;Saenz et al., 2010). The shared transposon tnpA-04 has been reported tobe highly expressed in manures, biosolids or amended soils (Li et al.,2017; Chen et al., 2016; Xie et al., 2016; Xu et al., 2016). This may beone of the most important MGEs involved in HGT in environmentalmedia. This phenomenon may explain how some ARGs are unique tospecific compartments. Different transposons or integrons mighttranspose only specific ARGs to host bacteria (Jones-Dias et al., 2016;Berendonk et al., 2015; Brooks et al., 2014).

Moreover, the tnpA gene increased significantly in soil with biosolidapplication and was a shared MGE in soil, root and shoot compart-ments. It seems that replicative transposition might play a leading rolein biosolid-driven HGT. tnpA gene protein can catalyze the synthesis ofcointegrate, a classic pattern of replicative transposition (Reed, 1981).Once the cointegrate resolves, the ARGs will be acquired by the re-ceptor and the original plasmid will not lose the transposons. The donorplasmid will retain the ability to transpose and this confers a potentiallyhigher probability of HGT occurring.

Although HGT might be less selective, it will be better mediated bycertain microbes. Lettuce consists of its own somatic cells as an un-damaged seed and colonization by environmental microbes will followas it develops and grows (Muller et al., 2016). LEfSe results at the 0.05probability level indicate that Actinobacteria and Proteobacteria werethe main phyla in plant endophytes and this agrees with previous stu-dies (Ulrich et al., 2008; Kuklinsky-Sobral et al., 2004; Sessitsch et al.,2002). Indeed, they are always found to be reservoirs of ARGs (Dantaset al., 2008; Li et al., 2006; Riesenfeld et al., 2004). This indicates aconsiderable risk of human exposure to ARGs through the recycling of

biosolids with their land application in food production although theyoccur at low abundance. Colonization by exogenous ARGs may lead to asudden outbreak (Manaia, 2017) and introducing ARGs into food pro-duction may prove to be foolhardy.

4.4. Visualizing the resistome using HT-qPCR

HT-qPCR was performed to provide a more comprehensive profileof ARG distribution at harvest. aadA1, acrB, msrC, tet(32)and VanRAwere found to heighten the risk of proliferation with biosolids appli-cation. Biosolids application may drive the shifts in the resistome indifferent compartments. Fig. 5 clearly shows that the expression ofsome ARGs, especially multidrug resistant genes, tended to be up-regulated in roots and shoots with biosolid application. Indeed, thetransmission ability of ARGs in the ecosystem might be an importantissue (Berendonk et al., 2015). However, they have not been found tobe important in previous studies using real-time PCR (Lau et al., 2017).

There were slight differences in bacterial taxa and ARG abundancesin soil at the 0.3% biosolid application rate and a distinct resistomeshowed in the control and 0.3% biosolid application treatment. HT-qPCR contributed to the finding that the other classes of ARGs reached30.9% while the gene resistance to aminoglycosides decreased by18.4%. A previous study concluded that there may be no risk fromARGs at a low biosolid application rate (4.5 t ha−1) judging from theabundance of ARGs (Chen et al., 2016) but the present study shows aneffect of biosolid application on the composition of ARGs even at a lowapplication rate (0.3%). This indicates that different classes of ARGs canbe distinguished according to their response to changes in the terrestrialenvironment. Further studies are required that attach importance to theevolution and dissemination of ARGs from the perspective of omics.

Fig. 6. Changes in OTU relative abundance at family level with different biosolid application rates. Changes in OTU relative abundance are determined by Css-C0,where Css is OTU relative abundance in soil with biosolid application and C0 is the OTU relative abundance in the control. * represents differences reachingsignificance level p < 0.05 by Student t-test.

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5. Conclusions

Biosolid application may increase the abundance of ARGs in soilsand lead to the accumulation of antibiotics. Accordingly, ARGs may betransferred into the food chain and threaten human health. Biosolid-borne ARGs account for the principal proportion of ARGs in soil re-sulting from biosolid application. Following biosolid application, bothinternal and external routes might contribute to the spread of ARGsfrom soil to lettuce at harvest. However, the ARGs of fresh edible lettucewill be mainly endogenous. Moreover, the transmission ability of ARGsfrom soil to lettuce changed with biosolid application. The presentstudy indicates that biosolid application may stimulate the transfer ofsome genes that confer multidrug resistance to the lettuce endophytes.Biosolid application drives the shift in ARGs mainly through horizontalgene transfer and this suggests higher risks of biosolid-borne ARGsspreading in the environment without selectivity. In the future thefeasibility of biosolid application must be evaluated from the perspec-tive of antibiotics and ARGs. Meanwhile, the abundance of ARGs shouldbe included in the control standards of biosolids for agricultural use.

Conflicts of interest

The authors declare no conflicts of interest.

Acknowledgments

This research was funded by the National Natural ScienceFoundation of China (41325003, 41401581) and the Science andTechnology Program of Jiangsu, China (BK20141049).

Appendix A. Supplementary data

Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.soilbio.2018.04.017.

References

AHI, 1997. Analysis of Data and Information to Support a PECsoil Trigger Value for Phase I(A Retrospective Review of Ecotoxicity Data from Environmental AssessmentsSubmitted to FDA/CVM to Support the Approval of Veterinary Drug Products in theUnited States from 1973-1997). USA. Animal Health Institute, Environmental RiskAssessment Working Group.

Andersson, D.I., Hughes, D., 2014. Microbiological effects of sublethal levels of anti-biotics. Nature Reviews Microbiology 12 (7), 465–478.

Aydin, S., Ince, B., Ince, O., 2015. Development of antibiotic resistance genes in microbialcommunities during long-term operation of anaerobic reactors in the treatment ofpharmaceutical wastewater. Water Research 83, 337–344.

Baker-Austin, C., Wright, M.S., Stepanauskas, R., McArthur, J.V., 2006. Co-selection ofantibiotic and metal resistance. Trends in Microbiology 14 (4), 176–182.

Berendonk, T.U., Manaia, C.M., Merlin, C., Fatta-Kassinos, D., Cytryn, E., Walsh, F.,Burgmann, H., Sorum, H., Norstrom, M., Pons, M.N., Kreuzinger, N., Huovinen, P.,Stefani, S., Schwartz, T., Kisand, V., Baquero, F., Martinez, J.L., 2015. Tackling an-tibiotic resistance: the environmental framework. Nature Reviews Microbiology 13(5), 310–317.

Bolger, A.M., Lohse, M., Usadel, B., 2014. Trimmomatic: a flexible trimmer for Illuminasequence data. Bioinformatics 30 (15), 2114–2120.

Brooks, J.P., Adeli, A., McLaughlin, M.R., 2014. Microbial ecology, bacterial pathogens,and antibiotic resistant genes in swine manure wastewater as influenced by threeswine management systems. Water Research 57, 96–103.

Caporaso, J.G., Kuczynski, J., Stombaugh, J., Bittinger, K., Bushman, F.D., Costello, E.K.,Fierer, N., Pena, A.G., Goodrich, J.K., Gordon, J.I., Huttley, G.A., Kelley, S.T.,Knights, D., Koenig, J.E., Ley, R.E., Lozupone, C.A., McDonald, D., Muegge, B.D.,Pirrung, M., Reeder, J., Sevinsky, J.R., Tumbaugh, P.J., Walters, W.A., Widmann, J.,Yatsunenko, T., Zaneveld, J., Knight, R., 2010. QIIME allows analysis of high-throughput community sequencing data. Nature Methods 7 (5), 335–336.

Chen, B.W., He, R., Yuan, K., Chen, E.Z., Lin, L., Chen, X., Sha, S., Zhong, J.A., Lin, L.,Yang, L.H., 2017a. Polycyclic aromatic hydrocarbons (PAHs) enriching antibioticresistance genes (ARGs) in the soils. Environmental Pollution 220, 1005–1013B.

Chen, Q.L., An, X.L., Li, H., Su, J.Q., Ma, Y.B., Zhu, Y.G., 2016. Long-term field appli-cation of sewage sludge increases the abundance of antibiotic resistance genes in soil.Environment International 92–93, 1–10.

Chen, Q.L., An, X.L., Zhu, Y.G., Su, J.Q., Gillings, M.R., Ye, Z.L., Cui, L., 2017b.Application of struvite alters the antibiotic resistome in soil, rhizosphere, and phyl-losphere. Environmental Science & Technology 51 (14), 8149–8157.

Chen, Q.L., An, X.L., Li, H., Zhu, Y.G., Su, J.Q., Cui, L., 2017c. Do manure-borne or in-digenous soil microorganisms influence the spread of antibiotic resistance genes inmanured soil? Soil Biology and Biochemistry 114, 229–237.

Cheng, M.M., Wu, L.H., Huang, Y.J., Luo, Y.M., Christie, P., 2014. Total concentrations ofheavy metals and occurrence of antibiotics in sewage sludges from cities throughoutChina. Journal of Soils and Sediments 14 (6), 1123–1135.

Dantas, G., Sommer, M.O.A., Oluwasegun, R.D., Church, G.M., 2008. Bacteria subsistingon antibiotics. Science 320 (5972), 100–103.

D'Costa, V.M., McGrann, K.M., Hughes, D.W., Wright, G.D., 2006. Sampling the antibioticresistome. Science 311 (5759), 374–377.

Edger, R.C., 2010. Search and clustering orders of magnitude faster than BLAST.Bioinformatics 26 (19), 2460–2461.

Heuer, H., Focks, A., Lamshoeft, M., Smalla, K., Matthies, M., Spiteller, M., 2008. Fate ofsulfadiazine administered to pigs and its quantitative effect on the dynamics ofbacterial resistance genes in manure and manured soil. Soil Biology and Biochemistry40 (7), 1892–1900.

Heuer, H., Kopmann, C., Binh, C.T.T., Top, E.M., Smalla, K., 2009. Spreading antibioticresistance through spread manure: characteristics of a novel plasmid type with low%G plus C content. Environmental Microbiology 11 (4), 937–949.

Hu, H.W., Wang, J.T., Li, J., Li, J.J., Ma, Y.B., Chen, D.L., He, J.Z., 2016. Field-basedevidence for copper contamination induced changes of antibiotic resistance in agri-cultural soils. Environmental Microbiology 18 (11), 3896–3909 (SI).

Hu, H.W., Wang, J.T., Li, J., Shi, X.Z., Ma, Y.B., Chen, D.L., He, J.Z., 2017. Long-termnickel contamination increases the occurrence of antibiotic resistance genes in agri-cultural soils. Environmental Science & Technology 51 (2), 790–800.

Huang, Y.J., Cheng, M.M., Li, W.H., Wu, L.H., Chen, Y.S., Luo, Y.M., Christie, P., Zhang,H.B., 2013. Simultaneous extraction of four classes of antibiotics in soil, manure andsewage sludge and analysis by liquid chromatography-tandem mass spectrometrywith the isotope-labelled internal standard method. Analytical Methods 5 (15),3721–3731.

Huerta, B., Marti, E., Gros, M., Lopez, P., Pompeo, M., Armengol, J., Barcelo, D., Balcazar,J.L., Rodriguez-Mozaz, S., Marce, R., 2013. Exploring the links between antibioticoccurrence, antibiotic resistance, and bacterial communities in water supply re-servoirs. The Science of the Total Environment 456, 161–170.

Jechalke, S., Kopmann, C., Rosendahl, I., Groeneweg, J., Weichelt, V., Krögerrecklenfort,E., Brandes, N., Nordwig, M., Ding, G.C., Siemens, J., Heuer, H., Smalla, K., 2013.Increased abundance and transferability of resistance genes after field application ofmanure from sulfadiazine-treated pigs. Applied and Environmental Microbiology 79,1704–1711.

Jones-Dias, D., Manageiro, V., Ferreira, E., Barreiro, P., Vieira, L., Moura, I.B., Caniça, M.,2016. Architecture of class 1, 2, and 3 integrons from gram negative bacteria re-covered among fruits and vegetables. Frontiers in Microbiology 7 (1400).

Kuklinsky-Sobral, J., Araujo, W.L., Mendes, R., Geraldi, I.O., Pizzirani-Kleiner, A.A.,Azevedo, J.L., 2004. Isolation and characterization of soybean-associated bacteriaand their potential for plant growth promotion. Environmental Microbiology 6 (12),1244–1251.

Laskaris, P., Tolba, S., Calvo-Bado, L., Wellington, L., 2010. Coevolution of antibioticproduction and counter-resistance in soil bacteria. Environmental Microbiology 12(3), 783–796.

Lau, C.H.F., Li, B., Zhang, T., Tien, Y.C., Scott, A., Murray, R., Sabourin, L., Lapen, D.R.,Duenk, P., Topp, E., 2017. Impact of pre-application treatment on municipal sludgecomposition, soil dynamics of antibiotic resistance genes, and abundance of anti-biotic-resistance genes on vegetables at harvest. The Science of the TotalEnvironment 587, 214–222.

Li, J.J., Xin, Z.H., Zhang, Y.Z., Chen, J.W., Yan, J.X., Li, H.J., Hu, H.W., 2017. Long-termmanure application increased the levels of antibiotics and antibiotic resistance genesin a greenhouse soil. Applied Soil Ecology 121, 193–200.

Li, W.B., Hartung, J.S., Levy, L., 2006. Quantitative real-time PCR for detection andidentification of Candidatus Liberibacter species associated with citrus huan-glongbing. Journal of Microbiological Methods 66 (1), 104–115.

Magoč, T., Salzberg, S.L., 2011. FLASH: fast length adjustment of short reads to improvegenome assemblies. Bioinformatics 27 (21), 2957–2963.

Manaia, C.M., 2017. Assessing the risk of antioiotic resistance transmission from theenvironment to humans: non-direct proportionality between abundance and risk.Trends in Microbiology 25 (3), 173–181.

Martínez, J.L., 2008. Antibiotics and antibiotic resistance genes in natural environments.Science 321 (5887), 365–367.

Martínez, J.L., Sanchez, M.B., Martinez-Solano, L., Hernandez, A., Garmendia, L.,Fajardo, A., Alvarez-Ortega, C., 2009. Functional role of bacterial multidrug effluxpumps in microbial natural ecosystems. FEMS Microbiology Reviews 33 (2),430–449.

McDonald, D., Price, M.N., Goodrich, J., Nawrocki, E.P., DeSantis, T.Z., Probst, A.,Andersen, G.L., Knight, R., Hugenholtz, P., 2012. An improved Greengenes taxonomywith explicit ranks for ecological and evolutionary analyses of bacteria and archaea.The ISME Journal 6 (3), 610–618.

Meng, X.Z., Venkatesan, A.K., Ni, Y.L., Steele, J.C., Wu, L.L., Bignert, A., Bergman, A.,Halden, R.U., 2016. Organic contaminants in Chinese sewage sludge: a meta-analysisof the literature of the past 30 years. Environmental Science & Technology 50 (11),5454–5466.

Michael, I., Rizzo, L., McArdell, C.S., Manaia, C.M., Merlin, C., Schwartz, T., Dagot, C.,Fatta-Kassinos, D., 2013. Urban wastewater treatment plants as hotspots for the re-lease of antibiotics in the environment: a review. Water Research 47 (3), 957–995.

Ministry of Environmental Protection of the People’s Republic of China, 1984. ControlStandards for Pollutants in Sludge from Agriculture Use, GB 4284–84. StandardsPress of China, Beijing.

Muller, D.B., Vogel, C., Bai, Y., Vorholt, J.A., 2016. The plant microbiota: systems-level

L. Yang et al. Soil Biology and Biochemistry 122 (2018) 131–140

139

insights and perspectives. Annual Review of Genetics 50, 211–234.Pu, C.J., Liu, H., Ding, G.C., Sun, Y., Yu, X.L., Chen, J.H., Ren, J.Y., Gong, X.Y., 2018.

Impact of direct application of biogas slurry and residue in fields: in situ analysis ofantibiotic resistance genes from pig manure to fields. Journal of Hazardous Materials344, 441–449.

Rahube, T.O., Marti, R., Scott, A., Tien, Y.C., Murray, R., Sabourin, L., Zhang, Y., Duenk,P., Lapen, D.R., Topp, E., 2013. Impact of fertilizing with raw or anaerobically di-gested sewage sludge on the abundance of antibiotic-resistant coliforms, antibioticresistance genes, and pathogenic bacteria in soil and on vegetables at harvest.Applied and Environmental Microbiology 80 (22), 6898–6907.

Rahube, T.O., Marti, R., Scott, A., Tien, Y.C., Murray, R., Sabourin, L., Duenk, P., Lapen,D.R., Topp, E., 2016. Persistence of antibiotic resistance and plasmid-associatedgenes in soil following application of sewage sludge and abundance on vegetables atharvest. Canadian Journal of Microbiology 62, 600–607.

Reed, R.R., 1981. Resolution of cointegrates between transposons gamma delta and Tn3defines the recombination site. Proceedings of the National Academy of Sciences 78(6), 3428–3432.

Riesenfeld, C.S., Goodman, R.M., Handelsman, J., 2004. Uncultured soil bacteria are areservoir of new antibiotic resistance genes. Environmental Microbiology 6 (9),981–989.

Rodriguez-Mozaz, S., Chamorro, S., Marti, E., Huerta, B., Gros, M., Sanchez-Melsio, A.,Borrego, C.M., Barcelo, D., Balcazar, J.L., 2015. Occurrence of antibiotics and anti-biotic resistance genes in hospital and urban wastewaters and their impact on thereceiving river. Water Research 69, 34–242.

Saenz, Y., Vinue, L., Ruiz, E., Somalo, S., Martinez, S., Rojo-Bezares, B., Zarazaga, M.,Torres, C., 2010. Class 1 integrons lacking qacE Delta 1 and sul1 genes in Escherichiacoli isolates of food, animal and human origins. Veterinary Microbiology 144 (3–4),493–497.

Segata, N., Izard, J., Waldron, L., Gevers, D., Miropolsky, L., Garrett, W.S., Huttenhower,C., 2011. Metagenomic biomarker discovery and explanation. Genome Biology 12(6), R60.

Sessitsch, A., Reiter, B., Pfeifer, U., Wilhelm, E., 2002. Cultivation-independent popula-tion analysis of bacterial endophytes in three potato varieties based on eubacterialand Actinomycetes-specific PCR of 16S rRNA genes. FEMS Microbiology Ecology 39(1), 23–32.

Sparks, D.L., Page, A.L., Helmke, P.A., Loeppert, R.H., Soltanpour, P.N., Tabatabai, M.A.,Johnston, C.T., Summer, M.E., 1996. Methods of Soil Analysis. Part 3. ChemicalMethods. 2Soil Science Society of America, Inc. American Society of Agronomy, Inc.,Madison, Wisconsin, USA.

Standardization Administration of the People’s Republic of China, 2009. Disposal ofSludge from Municipal Wastewater Treatment Plant-control Standards forAgricultural Use, CJ/T 309–2009. Standards Press of China, Beijing.

Stepanauskas, R., Glenn, T.C., Jagoe, C.H., Tuckfield, R.C., Lindell, A.H., McArthur, J.V.,2005. Elevated microbial tolerance to metals and antibiotics in metal-contaminated

industrial environments. Environmental Science & Technology 39 (10), 3671–3678.Su, J.Q., Wei, B., Ou-Yang, W.Y., Huang, F.Y., Zhao, Y., Xu, H.J., Zhu, Y.G., 2015.

Antibiotic resistome and its association with bacterial communities during sewagesludge composting. Environmental Science & Technology 49 (12), 7356–7363.

Ulrich, K., Ulrich, A., Ewald, D., 2008. Diversity of endophytic bacterial communities inpoplar grown under field conditions. FEMS Microbiology Ecology 63 (2), 169–180.

Wang, F.H., Qiao, M., Su, J.Q., Chen, Z., Zhou, X., Zhu, Y.G., 2014. High throughputprofiling of antibiotic resistance genes in urban park soils with reclaimed water ir-rigation. Environmental Science & Technology 48 (16), 9079–9085.

Wang, F.H., Qiao, M., Chen, Z., Su, J.Q., Zhu, Y.G., 2015. Antibiotic resistance genes inmanure-amended soil and vegetables at harvest. Journal of Hazardous Materials 299,215–221.

Wang, Q., Garrity, G.M., Tiedje, J.M., Cole, J.R., 2007. Naive Bayesian classifier for rapidassignment of rRNA sequences into the new bacterial taxonomy. Applied andEnvironmental Microbiology 73 (16), 5261–5267.

Wu, D., Huang, X.H., Sun, J.Z., Graham, D.W., Xie, B., 2017. Antibiotic resistance genesand associated microbial community conditions in aging landfill systems.Environmental Science & Technology 51 (21), 12859–12867.

Xie, W.Y., Yang, X.P., Li, Q., Wu, L.H., Shen, Q.R., Zhao, F.J., 2016. Changes in antibioticconcentrations and antibiotic resistome during commercial composting of animalmanures. Environmental Pollution 219, 182–190.

Xiong, W.G., Wang, Y.L., Sun, Y.X., Ma, L.P., Zeng, Q.L., Jiang, X.T., Li, A.D., Zeng, Z.L.,Zhang, T., 2018. Antibiotic-mediated changes in the fecal microbiome of broilerchickens define the incidence of antibiotic resistance genes. Microbiome 6, 34.

Xu, L.K., Ouyang, W.Y., Qian, Y.Y., Su, C., Su, J.Q., Chen, H., 2016. High-throughputprofiling of antibiotic resistance genes in drinking water treatment plants and dis-tribution systems. Environmental Pollution 213, 119–126.

Yang, G., Zhang, G.M., Wang, H.C., 2015. Current state of sludge production, manage-ment, treatment and disposal in China. Water Research 78, 60–73.

Yang, Q.X., Zhang, H., Guo, Y.H., Tian, T.T., 2016. Influence of chicken manure fertili-zation on antibiotic-resistant bacteria in soil and the endophytic bacteria of pakchoi.International Journal of Environmental Research and Public Health 13, 7.

Zhang, X.X., Zhang, T., 2011. Occurrence, abundance, and diversity of tetracycline re-sistance genes in 15 sewage treatment plants across China and other global locations.Environmental Science & Technology 45 (7), 2598–2604.

Zimmerman, N.B., Vitousek, P.M., 2012. Fungal endophyte communities reflect en-vironmental structuring across a Hawaiian landscape. Proceedings of the NationalAcademy of Sciences 109 (32), 13022–13027.

Zhou, T., Li, Z., Zhang, F., Jiang, X.S., Shi, W.M., Wu, L.H., Christie, P., 2016.Concentrations of arsenic, cadmium and lead in human hair and typical foods ineleven Chinese cities. Environmental Toxicology and Pharmacology 48, 150–156.

Zhu, B.K., Chen, Q.L., Cheng, S.C., Zhu, Y.G., 2017. Does organically produced lettuceharbor higher abundance of antibiotic resistance genes than conventionally pro-duced? Environment International 98, 152–159.

L. Yang et al. Soil Biology and Biochemistry 122 (2018) 131–140

140