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Environmental Pollution 241 (2018) 978e987

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Environmental Pollution

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Targeted inactivation of antibiotic-resistant Escherichia coli andPseudomonas aeruginosa in a soil-lettuce system by combinedpolyvalent bacteriophage and biochar treatment

Mao Ye a, Mingming Sun b, Yuanchao Zhao b, Wentao Jiao a, c, *, Bing Xia d, Manqiang Liu b,Yanfang Feng d, Zhongyun Zhang a, Dan Huang a, Rong Huang a, Jinzhong Wan e,Ruijun Du f, Xin Jiang a, **, Feng Hu b

a Key Laboratory of Soil Environment and Pollution Remediation, Institute of Soil Science, Chinese Academy of Sciences, Nanjing, 210008, Chinab Soil Ecology Lab, College of Resources and Environmental Sciences, Nanjing Agricultural University, Nanjing, 210095, Chinac State Key Laboratory of Urban and Regional Ecology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing, 100085, Chinad Anhui Academy of Environmental Science Research, Hefei, 230022, Chinae Institute of Agricultural Resources and Environment, Jiangsu Academy of Agricultural Sciences, Nanjing, 210014, Chinaf Nanjing Institute of Environmental Science, Ministry of Environmental Protection of China, Nanjing, 210042, China

a r t i c l e i n f o

Article history:Received 18 February 2018Received in revised form14 April 2018Accepted 16 April 2018

Keywords:Polyvalent bacteriophage therapyBiocharAntibiotic resistance genesEscherichia coli K-12Pseudomonas aeruginosa PAO1

* Corresponding author. Key Laboratory of SoilRemediation, Institute of Soil Science, Chinese Aca210008, China.** Corresponding author.

E-mail addresses: wtjiao@rcees.ac.cn (W. Jiao), jia

https://doi.org/10.1016/j.envpol.2018.04.0700269-7491/© 2018 Elsevier Ltd. All rights reserved.

a b s t r a c t

High abundances of antibiotic-resistant pathogenic bacteria (ARPB) and antibiotic resistance genes(ARGs) in agricultural soil-plant systems have become serious threats to human health and environ-mental safety. Therefore, it is crucial to develop targeted technology to control existing antibioticresistance (AR) contamination and potential dissemination in soil-plant systems. In this work, polyvalentbacteriophage (phage) therapy and biochar amendment were applied separately and in combination tostimulate ARPB/ARG dissipation in a soil-lettuce system. With combined application of biochar andpolyvalent phage, the abundance of Escherichia coli K-12 (tetR) and Pseudomonas aeruginosa PAO1(ampR þ fosR) and their corresponding ARGs (tetM, tetQ, tetW, ampC, and fosA) significantly decreased inthe soil after 63 days' incubation (p< 0.05). Similar results for endophytic K-12 and PAO1, and ARGs, werealso obtained in lettuce tissues following combined treatment. Additionally, high throughput sequencingrevealed that biochar and polyvalent phage synergetically improved the structural diversity and func-tional stability of the indigenous bacterial communities in soil and the endophytic ones in lettuce. Hence,this work proposes a novel biotechnology that combines biochar amendment and polyvalent phagetherapy to achieve targeted inactivation of ARPB, which stimulates ARG dissipation in soil-lettucesystems.

© 2018 Elsevier Ltd. All rights reserved.

1. Introduction

Due to the abuse of veterinary antibiotics and a lack of envi-ronmental management, antibiotic resistance (AR) contamina-tion has become a serious environmental concern at national andinternational scales (Burch et al., 2017; Chen et al., 2016). This isespecially crucial for agricultural soil near intensive livestock

Environment and Pollutiondemy of Sciences, Nanjing,

ngxin@issas.ac.cn (X. Jiang).

farming sites in China because of the application of livestockmanure with high contents of residual antibiotics and antibioticresistance genes (ARGs) (Liu et al., 2017; Peng et al., 2017; Xianget al., 2018). Meanwhile, the presence of large amounts of mobilegenetic elements (MGEs; plasmids, integrons, and transposons)has made soil environments hotspots of antibiotic-resistantbacteria (ARB) and ARGs (Leclercq et al., 2016; Sun et al., 2018;Yu et al., 2017a). Facilitated by horizontal gene transfer (HGT) andvertical transduction, the dissemination of enhanced antibiotic-resistant pathogenic bacteria (ARPB) has posed serious risks tothe health of humans and the environment (He et al., 2016; Jiaoet al., 2017; Yu et al., 2015). Therefore, there is an urgent need todevelop effective technology to decrease the risk of ARG

M. Ye et al. / Environmental Pollution 241 (2018) 978e987 979

proliferation caused by the novel biopollutants described above.Biochar has been widely accepted as an adsorbing material due

to its porous structure, high surface-area-to-volume ratio, andmicrobial hospitality (Duan et al., 2017; Vithanage et al., 2014). Inaddition, its capability of impeding ARB and ARG proliferation fromsoil to vegetables has been demonstrated by previous studies(Rajapaksha et al., 2015; Ye et al., 2016). By applying biochar to soiladjacent to a livestock farm, the abundance of ARB and ARGs wassignificantly decreased in both soil and vegetable samples (Cuiet al., 2017; Li et al., 2017). However, despite these overall de-clines, a fair amount of residual ARPB remained, with implicationsfor human health. As a consequence, furthermethods are needed toeliminate ARPB from soil-plant systems.

One of the most promising approaches is the use of phagetherapy to kill pathogens (Lyon, 2017; Petrovski et al., 2011; Pireset al., 2017). Bacteriophages (phages) are commonly considered tobe bacterial viruses that infect and replicate within specific hostbacteria (Pires et al., 2015; Yu et al., 2017b). They are the mostabundant living entities in the biosphere, with an estimated pop-ulation of 1031 (Keen et al., 2017; Khalifa et al., 2015). By applyingphage isolates to lysis host pathogens, phage therapy has shownpotential for application in a broad range of areas (De Smet et al.,2017; Drulis-Kawa et al., 2015). For instance, the lytic phageshave been used to reduce food-borne pathogens (Carvalho et al.,2017; Kazi and Annapure, 2016; Wei et al., 2017). In addition,with the recent recognition of the widespread prevalence of poly-valent phages in the environment, phage therapy has beenextended to eliminate ARPB in sludge during wastewater treatment(Amarillas et al., 2013; Gu et al., 2012; Hsieh et al., 2011; Mahmoudet al., 2018; Yu et al., 2017a). Yu et al. (2017b) reported the sup-pression of enteric bacteria by polyvalent phages, which therebydecreased residual ARPB abundance and proliferation risk in awastewater system. However, whether phage therapy can beapplied to reduce the levels of ARPB in soil environments remainsto be investigated. Also yet to be explored is the effect of combiningphage therapy and biochar application on APRB dissipation in soil-plant systems.

In this work, a microcosm experiment was set up to evaluate theimpact of biochar and polyvalent phage application on the dissi-pation of ARPB/ARG in a soil-lettuce system. The main objectiveswere to i) investigate the efficiency of polyvalent phages in elimi-nating two typical pathogenic bacteria (Escherichia coli K-12 andPseudomonas aeruginosa PAO1) in a soil-lettuce system; ii) examineARPB/ARG dissipation following biochar and polyvalent phageapplicationdboth separately and in combination; and iii) assessthe environmental impact and safety of the technique by moni-toring the compositions and structures of indigenous and endo-phytic bacterial communities in soil and lettuce tissues. This studyprovides insights into management practices to simultaneouslyinactivate ARPB and reduce ARG levels in soil-vegetable systems.

2. Materials and methods

2.1. Preparation of antibiotic-resistant pathogenic bacterial strains

Two host bacterial strains, Escherichia coli K-12 (ATCC 10798)and Pseudomonas aeruginosa PAO1 (ATCC 15692), were used in thiswork. Tryptone base layer agar and tryptone soft agar (TSA) wereused in the double-agar method. Both strains were incubated in atryptic soy broth medium at 37 �C overnight. Green fluorescentprotein marked E. coli K-12 bacteria carrying the plasmid-encodedtetM, tetQ, and tetW genes is resistant to tetracycline. Red fluores-cent protein marked Pseudomonas aeruginosa PAO1 carrying ampCand fosA genes in the chromosome is resistant to chloramphenicoland fosfomycin, respectively. The counts of K-12 and PAO1 were

measured by gfp and ref quantification (Nguyen et al., 2014;Zaborskyte et al., 2017).

2.2. Isolation of polyvalent phages against both K-12 and PAO1

Soil samples were collected in June 2017 at Zhu Jiashan dairyfarm, which has been operating for more than 20 years in Nanjing,Eastern China (31� 990 9000 N, 119� 100 400 E). Soil samples (10.0 g)were mixed with 10mL tryptic soy broth medium and incubatedovernight at 37 �C. Phages were separated from soil particles ac-cording to the sodium pyrophosphate method (Petrovski et al.,2011). Particles >0.2 mm were removed by centrifugation andfiltration (Yu et al., 2017a). The filtrate was further precipitated bypolyethylene glycol 8000 and resuspended in SM buffer to obtain aconcentrated phage stock (Drulis-Kawa et al., 2015). The phagestocks were then stored at 4 �C for subsequent polyvalent phageisolation within 5 days.

The sequential multihost isolation method was applied toisolate polyvalent phages against both K-12 and PAO1. K-12 samplesin an exponential growth phase were firstly mixed with the phagestock for 15min to allow phages to be adsorbed. Centrifugation(10,000 g, 5min) was then used to separate adsorbed and freephages. The supernatant was then mixed with K-12 for another15min to allow further phage adsorption. Phages obtained wereenriched afterwards with K-12 for 6 h. Then, the enriched phageswere mixed with PAO1 in an exponential phase (Hsieh et al., 2011;Yu et al., 2017b). The procedures described above were repeatedfive times.

In double-layer plate assays, plaques formed on the lawn ofPAO1 plates. Well-isolated plaques were cut from agar plates anddiluted with SM buffer. The phages were further purified fivetimes to remove contaminating phages according to standardprocedures (Kazi and Annapure, 2016; Khalifa et al., 2015).Transmission electron microscopy at 50 kV was used to identifythe morphological characteristics of obtained polyvalent phages(YSZ-1, Fig. S1) (Jebri et al., 2016). According to the InternationalCommittee on Taxonomy of Viruses guidelines, the YSZ-1 phagebelongs to the Podoviridae family (Tolstoy et al., 2018; Yutin et al.,2018). The YSZ-1 phage has a six-sided outline with an oblonghead of length 95 nm and width 70 nm, and a tubular tail with alength of 100 nm. It is a double-stranded lytic phage, with awhole genome of 61 kb. The optimal multiplicity of infection(MOI) for the host K-12 and PAO1 were the same, 1:10000, withlatent phases of 13 and 18min, respectively. The efficiency ofYSZ-1 in solely or simultaneously lysing K-12/PAO1 was ofmagnitude 3e4 (Fig. S2).

A DNA analysis was carried out according to the previousdescription (Sun et al., 2018). The YSZ-1 phage stock was firstconcentrated to 1mL (100 kDa Amicon Ultra centrifugal filter units,Millipore). DNase (100 U mL�1) was used to eliminate free DNAoutside the phage particles. TIANamp Virus DNA/RNA Kit (TIAN-GEN, Beijing, China) was then used to extract and purify phageDNA. Genes encoding Shiga stx1and stx2, enterotoxins ystA andystB, virulence factors virF, and adhesion yadA were not detected(Quir�os and Muniesa, 2017). Bacteriophages were stored at 4 �C inSM buffer for subsequent trials.

2.3. Soil microcosms and lettuce cultivation

Clean farmland soils were sampled in June 2017 at Guyu farm inthe suburban district of Nanjing, China (31� 660 9300 N, 118� 880 3700

E). Given that it is an organic farm, organic fertilizers containingantibiotics were not allowed to be applied. Clean soil samples(0e10 cm) were collected from 10 random locations around thefarm (about 100 kg in total). All soil samples were ground to pass

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through a 2.0mm sieve. The samples were composed of 19.7% sand,71.2% silt, and 9.1% clay. The soil had a pH of 6.8, 2.8% organic C,2.2 g kg�1 total N, and 0.29 g kg�1 total P. Antibiotics and ARGs werebelow the detection limit.

Artificial polluted soil was prepared by inoculation with K-12and PAO1 to obtain a final concentration of 108 CFUmL�1 in the soil.Soil microcosms were set up using a series of polyvinyl chloridecylindrical pots (bottom radius 10 cm; height 30 cm), each con-taining 8000 g of soil. The treatments were designated as follows:(A) CK: artificial polluted soil þ lettuce cultivation, (B) B: CK þ 0.5%(w/w) biochar amendment, (C) P: CK þ 104 (PFU g�1) YSZ-1 inoc-ulation, and (D) BP: CK þ 0.5% (w/w) biochar amendment þ 104

(PFU g�1) YSZ-1 inoculation. Besides the four treatments describedabove, six other treatments were also set as negative controls: (E)CS: negative control with clean soil; (F) PS: artificial polluted soil;(G) PSB: PS þ 0.5% (w/w) biochar amendment; (H) PSP: PS þ 104

(PFU/g) phage inoculation; (I) PSBP: CK þ 0.5% (w/w) biocharamendment þ 104 (PFU/g) phage inoculation; (J) SCL: negativecontrol with lettuce cultivation in clean soil. Triplicate sampleswere prepared. The biochar used here was maize straw pyrolyzedat 300e700 �C in a furnace in a pure N2 environment. It had a pH of9.8 and contained 423.2 g kg�1 ash, 483.2 g kg�1 total C, 2.1 g kg�1

total N, 113.2 g kg�1 K, 3.2 g kg�1 P, and 0.1 g kg�1 Si on a dry weightbasis. The specific surface area of the biochar was 262m2 g�1 (Yeet al., 2016).

Lettuce (Lactuca sativa L.) seeds were incubated for 7 days inmoist perlite. After that, three seedlings of similar sizewere plantedin each pot. The pots were incubated for 63 days in a greenhousechamber at 25± 2 �C. The initial soil moisture contents wereadjusted to 80% of maximum field capacity. Every other day,deionized water was added based on the weight loss of the pots.Every 10 days, approximately 10 g soil was collected by taking fiverandom soil samples up to a depth of 10 cm from each pot aroundthe lettuce roots. After 63 days of cultivation, soil and plant sampleswere stored in small plastic bags at 4 �C for subsequent trials. Pa-rameters, including the weight of lettuce tissues, total root surfacearea, root activity, chlorophyll content, and soluble protein content,were determined according to Ye et al. (2016).

2.4. K-12 and PAO1 in soil and lettuce analysis

Counts of K-12 and PAO1 in soil and lettuce tissues were con-ducted according to Section 2.1 (Nguyen et al., 2014; Zaborskyteet al., 2017). For the gfp labeled K-12 and rfp labeled PAO1 in let-tuce, the lettuce was first separated into roots and leaves. It wasthen washed thoroughly with sterilized physiological solution(8.5 g L�1 NaCl) to eliminate adhering particles and surface mi-crobes. The surface decontamination of the tissues was carried outaccording to Rahube et al. (2014). The leaves were immersed inhydrogen peroxide (30%, w/w) for 30min to eliminate the phyllo-sphere bacterial community, and thenwashedwith sterilized waterfour times. After that, the samples were treated with 70% ethanolfor 1min and washed in sterilized water again. Then tissue pieces(1 cm2) were homogenized and incubated at 28 �C in the dark for 5days on R2A agar medium (0.5 g L�1 proteose peptone, 0.5 g L�1

casamino acids, 0.5 g L�1 yeast extract, 0.5 g L�1 dextrose, 0.5 g L�1

soluble starch, 0.3 g L�1 dipotassium phosphate, 0.05 g L�1 mag-nesium sulfate$7H2O, 0.3 g L�1 sodium pyruvate, 15.0 g L�1 agar, pH7.4). Visible colonies of cultivable green and red bacterial endo-phytes on the R2A medium were counted. In addition, laser scan-ning confocal microscopy (Zeiss LSM710) was used to more directlydetect the distribution of K-12 and PAO1 in the lettuce (Nguyenet al., 2014; Zaborskyte et al., 2017).

2.5. ARG quantification in soil and lettuce

Quantification of ARG in the soil was determined according toour previous method (Ye et al., 2016; Sun et al., 2018). For ARGdetection in the lettuce, the root and leaf tissues were first sub-jected to surface decontamination. Fresh lettuce tissues (50 g ofroots/leaves) were mixed separately with 100mL sterile sodiummetaphosphate buffer (2.0 g L�1, pH 7.0) and macerated. Themaceratewas centrifuged at 12,000 rpm for 5min. Then, the pelletswere used for DNA extraction according to the same procedure asthat used for soil (Chen et al., 2016). Five ARGs (t tetM, tetQ, tetW,ampC and fosA) and eubacterial 16S rRNA genes were detected andquantified by quantitative PCR (qPCR) (He et al., 2016). All qPCRreactions were repeated three times. The primer design can befound in the Supporting Information, Table S1.

2.6. Changes of indigenous and endophytic bacterial biodiversity insoil

High-throughput sequencing technology was used to evaluatechanges in the diversity of indigenous and endophytic bacteria insoil. Soil DNA was extracted from 1.0 g samples using a Fast DNASPIN Kit for Soil (MP Biomedicals, CA) following the manufacturer'sinstructions. Lettuce tissues (5.0 g each of roots and leaves) weretransferred into a 50mL centrifuge tube and mixed with 45mLautoclaved 1� phosphate-buffered saline supplemented with0.02% Tween 20. The tubes were shaken at 200 rpm and 30 �C for2 h. After being filtered through a sterilized nylon net, the washsolution was centrifuged at 7500 rpm for 30min. The pellets werepreserved using the sodium phosphate buffer from the Fast DNASPIN Kit. The remaining procedure followed the Fast DNA SPIN Kitprotocol. The DNAwas assessed by 1.2% agarose gel electrophoresisand spectrophotometer analysis (Nano Drop ND-1000, Nano DropTechnologies, Willmington, DE) (He et al., 2016). Prepared DNAsamples were sent to TinyGene Co., Ltd. (Shanghai, China), and ashotgun library was constructed for each DNA sample. Then, Illu-mina high throughput sequencing with the HiSeq 2000 platformwas carried out employing a PE101 þ 8 þ 101 cycle sequencingstrategy. Subsequently, approximately 5 Gb of metagenomic datawere generated for each sample (Bates et al., 2013; Chen et al.,2016). Each DNA sample was carried out with three technicalreplicates.

2.7. Data analysis

Data from the experiments of using biochar as a soil amendmentand the impact of polyvalent phage therapy on K-12, PAO1 andARGs dissipation were analyzed by two-way ANOVA (SPSS 14.0software) and means were compared by least significant differ-ences (p< 0.05).

3. Results and discussion

3.1. Dynamic dissipation of ARPB and ARGs in soil

Soil is commonly accepted to be an important ARG and ARBreservoir in the environment, among which ARPB is of greaterimportance because of its close relevance to human health (Chenet al., 2016; Peng et al., 2017). Moreover, intensive antibiotic usein the last two decades has exerted selective pressure on AR bac-teria in soil and increased the risk of ARG dissemination throughthe soil food web (Burch et al., 2017; Liu et al., 2017). As a conse-quence, it is important to monitor ARG/ARPB fluctuations in the soilenvironment. As described in Fig. 1, the abundance of K-12 andPAO1 and their harbored ARGs was detected first. The counts of K-

Fig. 1. Dynamic abundances of K12 and PAO1 in soil in different treatments following 63 days cultivation. (A) CK: artificial polluted soil þ lettuce cultivation, (B) B: CK þ 0.5% (w/w)biochar amendment, (C) P: CK þ 104 (PFU/g) phage inoculation, and (D) BP: CK þ 0.5% (w/w) biochar amendment þ 104 (PFU/g) phage inoculation. Green triangle line: K12. Redcircular line: PAO1. Values are means± standard deviation of triplicate measurements. (For interpretation of the references to colour in this figure legend, the reader is referred tothe Web version of this article.)

M. Ye et al. / Environmental Pollution 241 (2018) 978e987 981

12 and PAO1 in the soil all declined clearly among the four treat-ments with lettuce cultivation (p< 0.05). In the soil with lettucecultivation, the dissipation of K-12wasmore significant than that ofPAO1. In contrast to the naturally-existing pathogenic bacteriumPAO1 in the soil, the K-12 used here was a gene-modified bacteria,which resulted in it having lower adaptability than that of PAO1(Nguyen et al., 2014; Zaborskyte et al., 2017). Therefore, it madesense that K-12 dissipated more quickly under the dual impacts ofbiochar and phage amendments.

Additionally, the dissipation of K-12 and PAO1 among treat-ments followed the order of BP > P > B > CK. Biochar as anenvironmentally-friendly soil conditioner, its application caneffectively increase the diversity and abundance of beneficialmicroorganisms in soil (Duan et al., 2017; Rajapaksha et al.,2015). Because of the increase in the ratio of beneficial bacteria,the K-12 and PAO1 both decreased clearly in the biocharamendment relative to the control (p < 0.05). In addition, thecounts of K-12 and PAO1 declined to magnitudes of 102 and103 CFU g�1 soil in the polyvalent phage inoculation treatment,suggesting it has a positive impact by simultaneously inactivatingmultiple host bacteria in the soil system. Compared to the singleamendment, two-way ANOVA analysis indicated that use ofbiochar and polyvalent phage together inactivates ARPB syn-ergetically (Table S2). For the combined treatment, a largeamount of bacteria tended to be adsorbed in/on the biochar,which improved the likelihood that the polyvalent phage couldattack the host bacteria (Vithanage et al., 2014; Ye et al., 2016).Consequently, the biochar and polyvalent phage worked syner-gistically to decrease pathogenic K-12 and PAO1 levels to agreater extent than either of the single treatments.

Meanwhile, four treatments without lettuce cultivation werealso investigated (Fig. S3). Similar to before, biochar and/or phageamendments significantly decreased the counts of K-12 and PAO1in the soil (Fig. S3, p< 0.05). However, compared to the treatmentswith lettuce cultivation, K-12 and PAO1 dissipation was muchgreater (Fig. 1, p< 0.05). Here, it was interesting to find that lettucecultivation slowed the attenuation of pathogens in the soil. Thiscould be caused by nutrient-rich root exudates (small molecularorganic acids, polysaccharide, and peptides, etc.) promoting thegrowth of indigenous microorganisms (Ye et al., 2016). As aconsequence, the nutrient input caused by lettuce cultivationclearly stimulated the abundance of pathogens, making them apersistent ecological threat in the soil.

Besides the pathogens, another emerging bio-pollutant whichcould disseminate along the food chain was ARGs in the soil.Therefore, the dynamics of ARG dissipation were also determinedin the present work. As exhibited in Figs. 2 and 3, S4, and S5, similardissipation dynamics were found for ARGs, K-12, and PAO1.Compared to the significant ARG dissipation that occurredfollowing the single amendment of biochar/phage, the combinedtreatment stimulated ARG attenuation to a greater extent, asdemonstrated by two-way ANOVA (Table S3). For the combinedtreatment, the levels of tetM, tetQ, tetW, ampC, and fosA declinedsignificantly from (1.6± 0.3)� 108, (2.1± 0.2)� 108, (2.7± 0.1)� 108,(5.7± 1.1)� 107, (9.4± 0.5)� 108 copies g�1 soil at day 1 to(1.2± 0.4)� 103, (9.1± 0.6)� 102, (7.2± 0.5)� 102, (5.4± 0.6)� 102,(9.1± 0.3)� 102 copies g�1 soil at day 63, respectively. Given that allthe ARGs detected here were harbored in the pathogens (K-12 orPAO1), it was reasonable that ARG abundance decreased when K-12and PAO1 were significantly lysed.

Fig. 2. Abundance of tetM, tetG and tetWgenes in soil before (Day 1) and after cultivation (Day 63) in different treatments. (A) CK: artificial polluted soil þ lettuce cultivation, (B) B:CK þ 0.5% (w/w) biochar amendment, (C) P: CK þ 104 (PFU/g) phage inoculation, and (D) BP: CK þ 0.5% (w/w) biochar amendment þ 104 (PFU/g) phage inoculation. Values aremeans ± standard deviation of triplicate measurements.

M. Ye et al. / Environmental Pollution 241 (2018) 978e987982

3.2. Control of ARPB and ARGs in lettuce tissues

The risk of soil ARG propagation is commonly increased by thefrequent passive/active transmission of ARB among various in-terfaces (soil, biological, air, and water systems) (Duan et al.,2017; Leclercq et al., 2016; Xiang et al., 2018). In soil and plantsystems, some ARPB in the soil can enter plant tissues andcolonize as endophytic bacteria (the so-called antibiotic resistantendophytic pathogenic bacteria, AREPB) (He et al., 2016; Jiaoet al., 2017; Ye et al., 2016). This process further enhances therisk of direct interaction between ARPB and human beings.Therefore, there is a new challenge to control ARPB/ARGs thattransfer from soil to colonize plant tissues. In this work, laserscanning confocal microscopy was used to monitor the distri-bution characteristics of K-12 and PAO1 in lettuce root and leaftissues among different treatments. As shown in Fig. 4, K-12(green dots) and PAO1 (red dots) counts in the root tissues wereclearly higher than those in the leaf tissues. Additionally, most ofthe bacteria were detected in the cavities between cells, espe-cially around the stomata of roots and leaves. Meanwhile, thebacterial counts in the root and leaf tissues followed the order ofCK > B > P > BP. The result obtained here suggests that biocharcan significantly impede the transmission of K-12/PAO1 from soilto lettuce tissues. Meanwhile, not only does polyvalent phageinactivate both K-12 and PAO1 in soil, it also decreases the countsof these strains in lettuce tissues, suggesting it has the ability tomove with and lyse the host bacteria, even inside the lettuce.

Moreover, the lowest K-12/PAO1 counts observed in the com-bined treatment demonstrated the additive effects of biochar andpolyvalent phage on decreasing ARPB dissemination risk in thesoil-lettuce system.

To quantitatively analyze the residual ARPB/ARGs in the let-tuce tissues, the abundance of K-12/PAO1 and ARGs weremeasured. As exhibited in Fig. 5, the total counts of K-12/PAO1were (5.8 ± 1.3)� 106 CFU g�1 in fresh roots and(4.1 ± 0.7)� 105 CFU g�1 in fresh leaves in control samples. It iswell known that lettuce is the most popular salad vegetable andcan be eaten raw. However, the AREPB detected in this studycannot be eliminated by current detergents since they colonizeinside the lettuce tissues. This supports our hypothesis that ARPBproliferation along the soil-vegetable-human being pathwayposes great threats to human health (He et al., 2016; Ye et al.,2016). Therefore, it was important to find that the applicationof biochar and/or polyvalent phage could significantly reduce thelevels of residual K-12/PAO1. The greatest dissipation wasobserved in the combined treatment, where the K-12/PAO1 countdecreased to (1.1 ± 0.2)� 104 CFU g�1 in fresh roots and(2.1 ± 0.2)� 103 CFU g�1 in fresh leaves (Fig. 5D). Then, theimpact of various treatments on lettuce ARG levels was investi-gated by qPCR. As shown in Figs. 6 and 7, the accumulativeabundance of five ARGs was (1.2 ± 0.7)� 108 copies g�1 in freshroots and (2.6 ± 0.4)� 106 copies g�1 in the fresh leaves of con-trols (Figs. 6A and 7A). For the combined treatment, however, theaccumulative level of ARGs declined significantly to

Fig. 3. Abundance of ampC and fosA genes in soil before (Day 1) and after cultivation (Day 63) in different treatments. (A) CK: artificial polluted soil þ lettuce cultivation, (B) B:CK þ 0.5% (w/w) biochar amendment, (C) P: CK þ 104 (PFU/g) phage inoculation, and (D) BP: CK þ 0.5% (w/w) biochar amendment þ 104 (PFU/g) phage inoculation. Values aremeans ± standard deviation of triplicate measurements.

Fig. 4. Confocal laser scanning microscope for the detection of K12 (green spot) and PAO1 (red spot) in lettuce roots (A, B, C, D) and leaves (E, F, G, H) in different treatments after 63days cultivation. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

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(5.8 ± 2.1)� 104 copies g�1 in fresh roots and (9.4 ± 3.1)� 103

copies g�1 in fresh leaves (Figs. 6D and 7D). Moreover, as theparameters of lettuce fresh weight, total root surface area, rootactivity, chlorophyll content, and soluble protein content(Table S4) were all enhanced, it is safe to conclude that thecombined treatment also improved lettuce quality.

3.3. Biodiversity of bacterial community in soil and lettuce

Evaluation of a technology like that presented in this paper liesnot only in its ability to remove pollutants but also on its influenceon the ecological environment (Chen et al., 2016; Sun et al., 2018).The polyvalent phage chosen here was a lytic phage that did not

Fig. 5. Counting of endophytic K12 and PAO1 in lettuce roots and leaves in different treatments. (A) CK: artificial polluted soil þ lettuce cultivation, (B) B: CK þ 0.5% (w/w) biocharamendment, (C) P: CK þ 104 (PFU/g) phage inoculation, and (D) BP: CK þ 0.5% (w/w) biochar amendment þ 104 (PFU/g) phage inoculation. Values are means ± standard deviation oftriplicate measurements.

Fig. 6. Abundance of tetM, tetG and tetW genes in lettuce before (Day 1) and after cultivation (Day 100) in different treatments. (A) CK: artificial polluted soil þ lettuce cultivation,(B) B: CK þ 0.5% (w/w) biochar amendment, (C) P: CK þ 104 (PFU/g) phage inoculation, and (D) BP: CK þ 0.5% (w/w) biochar amendment þ 104 (PFU/g) phage inoculation. Values aremeans ± standard deviation of triplicate measurements.

M. Ye et al. / Environmental Pollution 241 (2018) 978e987984

Fig. 7. Abundance of ampC and fosA genes in lettuce before (Day 1) and after cultivation (Day 100) in different treatments. (A) CK: artificial polluted soil þ lettuce cultivation, (B) B:CK þ 0.5% (w/w) biochar amendment, (C) P: CK þ 104 (PFU/g) phage inoculation, and (D) BP: CK þ 0.5% (w/w) biochar amendment þ 104 (PFU/g) phage inoculation. Values aremeans ± standard deviation of triplicate measurements.

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harbor the genes that encode shiga toxin, enterotoxin, virulentfactor, and adhesion toxin (Quir�os and Muniesa, 2017). Meanwhile,high throughput sequencing technology was used to evaluate theimpact of combined treatment on the diversity of soil indigenousand lettuce endophytic bacterial communities. It was found thatthe soil microbial diversity in the treatments with lettuce cultiva-tion (Fig. 8) was higher than those without cultivation (Fig. S6),suggesting a positive effect of root exudates on soil bacterial di-versity (Ye et al., 2016). As shown in Fig. 8, the total bacterial di-versity occurred in the order of soil> lettuce root> lettuce leaf,with more homologous bacteria detected between lettuce rootsand leaves. Additionally, the combined treatment obtained thehighest microbial diversity in soil and lettuce, following by biocharamendment, polyvalent phage inoculation, and controls. Previousresearch has reported the role of biochar in improving hydrother-mal ventilation and nutrient cycling in soil. Similarly, this work alsodemonstrated its use as an environmentally-friendly soil condi-tioner (Rajapaksha et al., 2015; Vithanage et al., 2014). When onlybiochar was applied, the soil bacterial diversity was enhancedsignificantly. However, endophytic bacterial diversity was signifi-cantly lower in lettuce tissues than in controls (Fig. 8, p< 0.05).Despite the fluctuation of bacterial diversity in soil and lettuce, theproportions of K-12 and PAO1 in soil and lettuce both decreaseddramatically to 2.4% and 2.9% compared to 4.0% and 8.8% in con-trols. In contrast to the biochar amendment, sole inoculation ofpolyvalent phage resulted in reduced diversities of soil and lettuceendophytic bacterial communities compared to controls (p< 0.05).This was likely caused by the polyvalent feature of the phage usedhere. Given that it could infect both K-12 and PAO1 and use them ashosts, it was quite possible for the phage to keep evolving anddevelop a broader range of hosts in the soil (Amarillas et al., 2013;Hsieh et al., 2011; Mahmoud et al., 2018). As the abundance ofmicroorganisms in the soil environment was much lower than that

occurring in themedium, it would be of great advantage to examinea broader range of hosts. Consequently, the diversity of the mi-crobial communities observed here could be reduced due to pre-dation (Gu et al., 2012; Yu et al., 2017a; b). However, when biocharand polyvalent phage were applied simultaneously, the diversity ofbacterial communities was restored compared to controls(p< 0.05). In addition, a higher diversity of beneficial bacteria wasfound in the treatments with biochar addition (B and BP treatment)compared to controls, including carbon-transforming bacteria(Bacillus, Pedomicrobium, and Pedobacter), nitrogen-fixing bacteria(Burkholderia, Azoarcus, Rhizobium, Nocardioides, and Meso-rhizobium), phosphorus-transforming bacteria (Paraclostridium),and sulfur-transforming facilitating bacteria (Sulfobacillus, Bdello-vibrio, and Cellvibrio). This further demonstrates the positive role ofbiochar in enhancing the biological functioning of microbial com-munities in the soil (Cui et al., 2017; Li et al., 2017; Rajapaksha et al.,2015). Meanwhile, the counts of bacteria, fungi, and actinomyces,and MBC and MBN activities increased most significantly in thecombined treatment (Table S5). Therefore, it is safe to conclude thatthe technique combining biochar and polyvalent phage not onlysignificantly decreased the environmental risk by dissipating ARPB/ARGs, but also restored the biological functioning of microbialcommunities in the soil-lettuce system. Meanwhile, consideringthe complex types of contaminated soil that exist, it is essential tocarry out further research to apply the combined technique to theremediation of ARG/ARPB contaminated soils.

4. Conclusions

A novel biotechnology combining biochar and polyvalent phagewas developed in this study to stimulate the dissipation ofantibiotic-resistant pathogens in a soil-lettuce system. After 63days of incubation, the dissemination of E. coli K-12 (tetR) and

Fig. 8. Relative abundance of bacterial population at genus level in soil (A, B, C, D), lettuce roots (E, F, G, H) and leaves (I, J, K, L) from different treatments with high-throughputsequencing technology. CK: artificial polluted soil þ lettuce cultivation, B: CK þ 0.5% (w/w) biochar amendment, P: CK þ 104 (PFU/g) phage inoculation, and BP: CK þ 0.5% (w/w)biochar amendment þ 104 (PFU/g) phage inoculation.

M. Ye et al. / Environmental Pollution 241 (2018) 978e987986

P. aeruginosa PAO1 (ampCR þ fosAR) was significantly impeded fromsoil to lettuce tissues. In addition, ARG abundance in the soil-lettucesystem declined clearly. High throughput sequencing technologyanalysis indicated the positive effect of the combined treatment onthe restoration of the microbial communities in the soil system.This work provides novel information regarding the targetedinactivation of multiple ARPB and ARGs, therein controlling theirdissemination risks in soil-vegetable-human systems.

Acknowledgement

This work was financially supported by the EnvironmentalProtection Research Project in Jiangsu Provincial EnvironmentalDepartment (2017005), the Jiangsu Agricultural Science and Tech-nology Innovation Fund (CX(17)3047), the Scientific ApparatusResearch and Development Program of Chinese Academy of Sci-ences (Young Talents: YJKYYQ20170057), the Jiangsu MunicipalNatural Science Foundation (BK20141050), the Leading Project ofthe Institute of Soil Science, Chinese Academy of Sciences (ISSA-SIP1655), the National Natural Science Foundation of China grants

(41771350), the Fundamental Research Funds for the Central Uni-versities (Y0201700160).

Appendix A. Supplementary data

Supplementary data related to this article can be found athttps://doi.org/10.1016/j.envpol.2018.04.070.

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