Environmental Pollution 241 (2018) 978e987

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Environmental Pollution
journal homepage: www.elsevier.com/locate/envpol

Targeted inactivation of antibiotic-resistant Escherichia coli and

Pseudomonas aeruginosa in a soil-lettuce system by combined
polyvalent 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, China
b
Soil Ecology Lab, College of Resources and Environmental Sciences, Nanjing Agricultural University, Nanjing, 210095, China
c
State Key Laboratory of Urban and Regional Ecology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing, 100085, China
d
Anhui Academy of Environmental Science Research, Hefei, 230022, China
e
Institute of Agricultural Resources and Environment, Jiangsu Academy of Agricultural Sciences, Nanjing, 210014, China
f
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 a b s t r a c t

Article history: High abundances of antibiotic-resistant pathogenic bacteria (ARPB) and antibiotic resistance genes
Received 18 February 2018 (ARGs) in agricultural soil-plant systems have become serious threats to human health and environ-
Received in revised form mental safety. Therefore, it is crucial to develop targeted technology to control existing antibiotic
14 April 2018
resistance (AR) contamination and potential dissemination in soil-plant systems. In this work, polyvalent
Accepted 16 April 2018
Available online 19 June 2018
bacteriophage (phage) therapy and biochar amendment were applied separately and in combination to
stimulate ARPB/ARG dissipation in a soil-lettuce system. With combined application of biochar and
polyvalent phage, the abundance of Escherichia coli K-12 (tetR) and Pseudomonas aeruginosa PAO1
Keywords:
Polyvalent bacteriophage therapy
(ampR þ fosR) and their corresponding ARGs (tetM, tetQ, tetW, ampC, and fosA) significantly decreased in
Biochar the soil after 63 days' incubation (p < 0.05). Similar results for endophytic K-12 and PAO1, and ARGs, were
Antibiotic resistance genes also obtained in lettuce tissues following combined treatment. Additionally, high throughput sequencing
Escherichia coli K-12 revealed that biochar and polyvalent phage synergetically improved the structural diversity and func-
Pseudomonas aeruginosa PAO1 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 phage
therapy to achieve targeted inactivation of ARPB, which stimulates ARG dissipation in soil-lettuce
systems.
© 2018 Elsevier Ltd. All rights reserved.

1. Introduction farming sites in China because of the application of livestock

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 and
international scales (Burch et al., 2017; Chen et al., 2016). This is
especially crucial for agricultural soil near intensive livestock
* Corresponding author. Key Laboratory of Soil Environment and Pollution
Remediation, Institute of Soil Science, Chinese Academy of Sciences, Nanjing,
210008, China.
** Corresponding author.
E-mail addresses: wtjiao@rcees.ac.cn (W. Jiao), jiangxin@issas.ac.cn (X. Jiang).
manure with high contents of residual antibiotics and antibiotic
resistance genes (ARGs) (Liu et al., 2017; Peng et al., 2017; Xiang
et al., 2018). Meanwhile, the presence of large amounts of mobile
genetic elements (MGEs; plasmids, integrons, and transposons)
has made soil environments hotspots of antibiotic-resistant
bacteria (ARB) and ARGs (Leclercq et al., 2016; Sun et al., 2018;
Yu et al., 2017a). Facilitated by horizontal gene transfer (HGT) and
vertical transduction, the dissemination of enhanced antibiotic-
resistant pathogenic bacteria (ARPB) has posed serious risks to
the health of humans and the environment (He et al., 2016; Jiao
et al., 2017; Yu et al., 2015). Therefore, there is an urgent need to
develop effective technology to decrease the risk of ARG

https://doi.org/10.1016/j.envpol.2018.04.070
0269-7491/© 2018 Elsevier Ltd. All rights reserved.
 M. Ye et al. / Environmental Pollution 241 (2018) 978e987
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, and
microbial hospitality (Duan et al., 2017; Vithanage et al., 2014). In
addition, its capability of impeding ARB and ARG proliferation from
soil to vegetables has been demonstrated by previous studies
(Rajapaksha et al., 2015; Ye et al., 2016). By applying biochar to soil
adjacent to a livestock farm, the abundance of ARB and ARGs was
significantly decreased in both soil and vegetable samples (Cui
et al., 2017; Li et al., 2017). However, despite these overall de-
clines, a fair amount of residual ARPB remained, with implications
for human health. As a consequence, further methods are needed to
eliminate ARPB from soil-plant systems.
One of the most promising approaches is the use of phage
therapy to kill pathogens (Lyon, 2017; Petrovski et al., 2011; Pires
et al., 2017). Bacteriophages (phages) are commonly considered to
be bacterial viruses that infect and replicate within specific host
bacteria (Pires et al., 2015; Yu et al., 2017b). They are the most
abundant living entities in the biosphere, with an estimated pop-
ulation of 1031 (Keen et al., 2017; Khalifa et al., 2015). By applying
phage isolates to lysis host pathogens, phage therapy has shown
potential for application in a broad range of areas (De Smet et al.,
2017; Drulis-Kawa et al., 2015). For instance, the lytic phages
have 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 been
extended to eliminate ARPB in sludge during wastewater treatment
(Amarillas et al., 2013; Gu et al., 2012; Hsieh et al., 2011; Mahmoud
et al., 2018; Yu et al., 2017a). Yu et al. (2017b) reported the sup-
pression of enteric bacteria by polyvalent phages, which thereby
decreased residual ARPB abundance and proliferation risk in a
wastewater system. However, whether phage therapy can be
applied to reduce the levels of ARPB in soil environments remains
to be investigated. Also yet to be explored is the effect of combining
phage therapy and biochar application on APRB dissipation in soil-
plant systems.
In this work, a microcosm experiment was set up to evaluate the
impact of biochar and polyvalent phage application on the dissi-
pation of ARPB/ARG in a soil-lettuce system. The main objectives
were to i) investigate the efficiency of polyvalent phages in elimi-
nating two typical pathogenic bacteria (Escherichia coli K-12 and
Pseudomonas aeruginosa PAO1) in a soil-lettuce system; ii) examine
ARPB/ARG dissipation following biochar and polyvalent phage
applicationdboth separately and in combination; and iii) assess
the 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 study
provides insights into management practices to simultaneously
inactivate 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 this
work. Tryptone base layer agar and tryptone soft agar (TSA) were
used in the double-agar method. Both strains were incubated in a
tryptic soy broth medium at 37  C overnight. Green fluorescent
protein marked E. coli K-12 bacteria carrying the plasmid-encoded
tetM, tetQ, and tetW genes is resistant to tetracycline. Red fluores-
cent protein marked Pseudomonas aeruginosa PAO1 carrying ampC
and fosA genes in the chromosome is resistant to chloramphenicol
and fosfomycin, respectively. The counts of K-12 and PAO1 were
979
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 dairy
farm, 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 10 mL tryptic soy broth medium and incubated
overnight 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 and
filtration (Yu et al., 2017a). The filtrate was further precipitated by
polyethylene glycol 8000 and resuspended in SM buffer to obtain a
concentrated phage stock (Drulis-Kawa et al., 2015). The phage
stocks were then stored at 4  C for subsequent polyvalent phage
isolation within 5 days.
The sequential multihost isolation method was applied to
isolate polyvalent phages against both K-12 and PAO1. K-12 samples
in an exponential growth phase were firstly mixed with the phage
stock for 15 min to allow phages to be adsorbed. Centrifugation
(10,000 g, 5 min) was then used to separate adsorbed and free
phages. The supernatant was then mixed with K-12 for another
15 min to allow further phage adsorption. Phages obtained were
enriched afterwards with K-12 for 6 h. Then, the enriched phages
were mixed with PAO1 in an exponential phase (Hsieh et al., 2011;
Yu et al., 2017b). The procedures described above were repeated
five times.
In double-layer plate assays, plaques formed on the lawn of
PAO1 plates. Well-isolated plaques were cut from agar plates and
diluted with SM buffer. The phages were further purified five
times to remove contaminating phages according to standard
procedures (Kazi and Annapure, 2016; Khalifa et al., 2015).
Transmission electron microscopy at 50 kV was used to identify
the morphological characteristics of obtained polyvalent phages
(YSZ-1, Fig. S1) (Jebri et al., 2016). According to the International
Committee on Taxonomy of Viruses guidelines, the YSZ-1 phage
belongs to the Podoviridae family (Tolstoy et al., 2018; Yutin et al.,
2018). The YSZ-1 phage has a six-sided outline with an oblong
head of length 95 nm and width 70 nm, and a tubular tail with a
length of 100 nm. It is a double-stranded lytic phage, with a
whole genome of 61 kb. The optimal multiplicity of infection
(MOI) for the host K-12 and PAO1 were the same, 1:10000, with
latent phases of 13 and 18 min, respectively. The efficiency of
YSZ-1 in solely or simultaneously lysing K-12/PAO1 was of
magnitude 3e4 (Fig. S2).
A DNA analysis was carried out according to the previous
description (Sun et al., 2018). The YSZ-1 phage stock was first
concentrated to 1 mL (100 kDa Amicon Ultra centrifugal filter units,
Millipore). DNase (100 U mL1) was used to eliminate free DNA
outside the phage particles. TIANamp Virus DNA/RNA Kit (TIAN-
GEN, Beijing, China) was then used to extract and purify phage
DNA. Genes encoding Shiga stx1and stx2, enterotoxins ystA and
ystB, virulence factors virF, and adhesion yadA were not detected
(Quiro
s and Muniesa, 2017). Bacteriophages were stored at 4  C in
SM buffer for subsequent trials.
2.3. Soil microcosms and lettuce cultivation
Clean farmland soils were sampled in June 2017 at Guyu farm in
the suburban district of Nanjing, China (31 660 9300 N, 118 880 3700
E). Given that it is an organic farm, organic fertilizers containing
antibiotics were not allowed to be applied. Clean soil samples
(0e10 cm) were collected from 10 random locations around the
farm (about 100 kg in total). All soil samples were ground to pass
980 M. Ye et al. / Environmental Pollution 241 (2018) 978e987
through a 2.0 mm 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 kg1 total N, and 0.29 g kg1 total P. Antibiotics and ARGs were
below the detection limit.
Artificial polluted soil was prepared by inoculation with K-12
and PAO1 to obtain a final concentration of 108 CFU mL1 in the soil.
Soil microcosms were set up using a series of polyvinyl chloride
cylindrical 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 g1) YSZ-1 inoc-
ulation, and (D) BP: CK þ 0.5% (w/w) biochar amendment þ 104
(PFU g1) YSZ-1 inoculation. Besides the four treatments described
above, 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) biochar
amendment þ 104 (PFU/g) phage inoculation; (J) SCL: negative
control with lettuce cultivation in clean soil. Triplicate samples
were prepared. The biochar used here was maize straw pyrolyzed
at 300e700  C in a furnace in a pure N2 environment. It had a pH of
9.8 and contained 423.2 g kg1 ash, 483.2 g kg1 total C, 2.1 g kg1
total N, 113.2 g kg1 K, 3.2 g kg1 P, and 0.1 g kg1 Si on a dry weight
basis. The specific surface area of the biochar was 262 m2 g1 (Ye
et al., 2016).
Lettuce (Lactuca sativa L.) seeds were incubated for 7 days in
moist perlite. After that, three seedlings of similar size were planted
in each pot. The pots were incubated for 63 days in a greenhouse
chamber at 25 ± 2  C. The initial soil moisture contents were
adjusted 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 five
random soil samples up to a depth of 10 cm from each pot around
the lettuce roots. After 63 days of cultivation, soil and plant samples
were stored in small plastic bags at 4  C for subsequent trials. Pa-
rameters, including the weight of lettuce tissues, total root surface
area, 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; Zaborskyte
et 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 was
then washed thoroughly with sterilized physiological solution
(8.5 g L1 NaCl) to eliminate adhering particles and surface mi-
crobes. The surface decontamination of the tissues was carried out
according to Rahube et al. (2014). The leaves were immersed in
hydrogen peroxide (30%, w/w) for 30 min to eliminate the phyllo-
sphere bacterial community, and then washed with sterilized water
four times. After that, the samples were treated with 70% ethanol
for 1 min and washed in sterilized water again. Then tissue pieces
(1 cm2) were homogenized and incubated at 28  C in the dark for 5
days on R2A agar medium (0.5 g L1 proteose peptone, 0.5 g L1
casamino acids, 0.5 g L1 yeast extract, 0.5 g L1 dextrose, 0.5 g L1
soluble starch, 0.3 g L1 dipotassium phosphate, 0.05 g L1 mag-
nesium sulfate$7H2O, 0.3 g L1 sodium pyruvate, 15.0 g L1 agar, pH
7.4). 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 directly
detect the distribution of K-12 and PAO1 in the lettuce (Nguyen
et al., 2014; Zaborskyte et al., 2017).
2.5. ARG quantification in soil and lettuce
Quantification of ARG in the soil was determined according to
our previous method (Ye et al., 2016; Sun et al., 2018). For ARG
detection in the lettuce, the root and leaf tissues were first sub-
jected to surface decontamination. Fresh lettuce tissues (50 g of
roots/leaves) were mixed separately with 100 mL sterile sodium
metaphosphate buffer (2.0 g L1, pH 7.0) and macerated. The
macerate was centrifuged at 12,000 rpm for 5 min. Then, the pellets
were used for DNA extraction according to the same procedure as
that used for soil (Chen et al., 2016). Five ARGs (t tetM, tetQ, tetW,
ampC and fosA) and eubacterial 16S rRNA genes were detected and
quantified by quantitative PCR (qPCR) (He et al., 2016). All qPCR
reactions were repeated three times. The primer design can be
found in the Supporting Information, Table S1.
2.6. Changes of indigenous and endophytic bacterial biodiversity in
soil
High-throughput sequencing technology was used to evaluate
changes in the diversity of indigenous and endophytic bacteria in
soil. Soil DNA was extracted from 1.0 g samples using a Fast DNA
SPIN Kit for Soil (MP Biomedicals, CA) following the manufacturer's
instructions. Lettuce tissues (5.0 g each of roots and leaves) were
transferred into a 50 mL centrifuge tube and mixed with 45 mL
autoclaved 1  phosphate-buffered saline supplemented with
0.02% Tween 20. The tubes were shaken at 200 rpm and 30  C for
2 h. After being filtered through a sterilized nylon net, the wash
solution was centrifuged at 7500 rpm for 30 min. The pellets were
preserved using the sodium phosphate buffer from the Fast DNA
SPIN Kit. The remaining procedure followed the Fast DNA SPIN Kit
protocol. The DNA was assessed by 1.2% agarose gel electrophoresis
and spectrophotometer analysis (Nano Drop ND-1000, Nano Drop
Technologies, Willmington, DE) (He et al., 2016). Prepared DNA
samples were sent to TinyGene Co., Ltd. (Shanghai, China), and a
shotgun library was constructed for each DNA sample. Then, Illu-
mina high throughput sequencing with the HiSeq 2000 platform
was carried out employing a PE101 þ 8 þ 101 cycle sequencing
strategy. Subsequently, approximately 5 Gb of metagenomic data
were generated for each sample (Bates et al., 2013; Chen et al.,
2016). Each DNA sample was carried out with three technical
replicates.
2.7. Data analysis
Data from the experiments of using biochar as a soil amendment
and the impact of polyvalent phage therapy on K-12, PAO1 and
ARGs dissipation were analyzed by two-way ANOVA (SPSS 14.0
software) 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 ARB
reservoir in the environment, among which ARPB is of greater
importance because of its close relevance to human health (Chen
et al., 2016; Peng et al., 2017). Moreover, intensive antibiotic use
in the last two decades has exerted selective pressure on AR bac-
teria in soil and increased the risk of ARG dissemination through
the 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 soil
environment. As described in Fig. 1, the abundance of K-12 and
PAO1 and their harbored ARGs was detected first. The counts of K-
 M. Ye et al. / Environmental Pollution 241 (2018) 978e987 981

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. Red
circular 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 to
the Web version of this article.)

12 and PAO1 in the soil all declined clearly among the four treat-
ments with lettuce cultivation (p < 0.05). In the soil with lettuce
cultivation, the dissipation of K-12 was more significant than that of
PAO1. In contrast to the naturally-existing pathogenic bacterium
PAO1 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 made
sense that K-12 dissipated more quickly under the dual impacts of
biochar and phage amendments.
Additionally, the dissipation of K-12 and PAO1 among treat-
ments followed the order of BP > P > B > CK. Biochar as an
environmentally-friendly soil conditioner, its application can
effectively increase the diversity and abundance of beneficial
microorganisms 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 biochar
amendment relative to the control (p < 0.05). In addition, the
counts of K-12 and PAO1 declined to magnitudes of 102 and
103 CFU g1 soil in the polyvalent phage inoculation treatment,
suggesting it has a positive impact by simultaneously inactivating
multiple host bacteria in the soil system. Compared to the single
amendment, two-way ANOVA analysis indicated that use of
biochar and polyvalent phage together inactivates ARPB syn-
ergetically (Table S2). For the combined treatment, a large
amount of bacteria tended to be adsorbed in/on the biochar,
which improved the likelihood that the polyvalent phage could
attack 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 a
greater extent than either of the single treatments.
Meanwhile, four treatments without lettuce cultivation were
also investigated (Fig. S3). Similar to before, biochar and/or phage
amendments significantly decreased the counts of K-12 and PAO1
in the soil (Fig. S3, p < 0.05). However, compared to the treatments
with lettuce cultivation, K-12 and PAO1 dissipation was much
greater (Fig. 1, p < 0.05). Here, it was interesting to find that lettuce
cultivation slowed the attenuation of pathogens in the soil. This
could be caused by nutrient-rich root exudates (small molecular
organic acids, polysaccharide, and peptides, etc.) promoting the
growth of indigenous microorganisms (Ye et al., 2016). As a
consequence, the nutrient input caused by lettuce cultivation
clearly stimulated the abundance of pathogens, making them a
persistent ecological threat in the soil.
Besides the pathogens, another emerging bio-pollutant which
could disseminate along the food chain was ARGs in the soil.
Therefore, the dynamics of ARG dissipation were also determined
in the present work. As exhibited in Figs. 2 and 3, S4, and S5, similar
dissipation dynamics were found for ARGs, K-12, and PAO1.
Compared to the significant ARG dissipation that occurred
following the single amendment of biochar/phage, the combined
treatment stimulated ARG attenuation to a greater extent, as
demonstrated by two-way ANOVA (Table S3). For the combined
treatment, the levels of tetM, tetQ, tetW, ampC, and fosA declined
significantly 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 g1 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 g1 soil at day 63, respectively. Given that all
the ARGs detected here were harbored in the pathogens (K-12 or
PAO1), it was reasonable that ARG abundance decreased when K-12
and PAO1 were significantly lysed.
982 M. Ye et al. / Environmental Pollution 241 (2018) 978e987
Fig. 2. Abundance of tetM, tetG and tetW 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 are
means ± standard deviation of triplicate measurements.
3.2. Control of ARPB and ARGs in lettuce tissues
The risk of soil ARG propagation is commonly increased by the
frequent 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 plant
systems, some ARPB in the soil can enter plant tissues and
colonize as endophytic bacteria (the so-called antibiotic resistant
endophytic pathogenic bacteria, AREPB) (He et al., 2016; Jiao
et al., 2017; Ye et al., 2016). This process further enhances the
risk of direct interaction between ARPB and human beings.
Therefore, there is a new challenge to control ARPB/ARGs that
transfer from soil to colonize plant tissues. In this work, laser
scanning confocal microscopy was used to monitor the distri-
bution characteristics of K-12 and PAO1 in lettuce root and leaf
tissues among different treatments. As shown in Fig. 4, K-12
(green dots) and PAO1 (red dots) counts in the root tissues were
clearly higher than those in the leaf tissues. Additionally, most of
the bacteria were detected in the cavities between cells, espe-
cially around the stomata of roots and leaves. Meanwhile, the
bacterial counts in the root and leaf tissues followed the order of
CK > B > P > BP. The result obtained here suggests that biochar
can significantly impede the transmission of K-12/PAO1 from soil
to lettuce tissues. Meanwhile, not only does polyvalent phage
inactivate both K-12 and PAO1 in soil, it also decreases the counts
of these strains in lettuce tissues, suggesting it has the ability to
move 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 and
polyvalent phage on decreasing ARPB dissemination risk in the
soil-lettuce system.
To quantitatively analyze the residual ARPB/ARGs in the let-
tuce tissues, the abundance of K-12/PAO1 and ARGs were
measured. As exhibited in Fig. 5, the total counts of K-12/PAO1
were (5.8 ± 1.3)  106 CFU g1 in fresh roots and
(4.1 ± 0.7)  105 CFU g1 in fresh leaves in control samples. It is
well known that lettuce is the most popular salad vegetable and
can be eaten raw. However, the AREPB detected in this study
cannot be eliminated by current detergents since they colonize
inside the lettuce tissues. This supports our hypothesis that ARPB
proliferation along the soil-vegetable-human being pathway
poses great threats to human health (He et al., 2016; Ye et al.,
2016). Therefore, it was important to find that the application
of biochar and/or polyvalent phage could significantly reduce the
levels of residual K-12/PAO1. The greatest dissipation was
observed in the combined treatment, where the K-12/PAO1 count
decreased to (1.1 ± 0.2)  104 CFU g1 in fresh roots and
(2.1 ± 0.2)  103 CFU g1 in fresh leaves (Fig. 5D). Then, the
impact of various treatments on lettuce ARG levels was investi-
gated by qPCR. As shown in Figs. 6 and 7, the accumulative
abundance of five ARGs was (1.2 ± 0.7)  108 copies g1 in fresh
roots and (2.6 ± 0.4)  106 copies g1 in the fresh leaves of con-
trols (Figs. 6A and 7A). For the combined treatment, however, the
accumulative level of ARGs declined significantly to
 M. Ye et al. / Environmental Pollution 241 (2018) 978e987 983

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 are
means ± 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 63
days cultivation. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

(5.8 ± 2.1)  104 copies g1 in fresh roots and (9.4 ± 3.1)  103
copies g1 in fresh leaves (Figs. 6D and 7D). Moreover, as the
parameters of lettuce fresh weight, total root surface area, root
activity, chlorophyll content, and soluble protein content
(Table S4) were all enhanced, it is safe to conclude that the
combined 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 lies
not only in its ability to remove pollutants but also on its influence
on the ecological environment (Chen et al., 2016; Sun et al., 2018).
The polyvalent phage chosen here was a lytic phage that did not
984 M. Ye et al. / Environmental Pollution 241 (2018) 978e987

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) 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 are means ± standard deviation of
triplicate 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 are
means ± standard deviation of triplicate measurements.
 M. Ye et al. / Environmental Pollution 241 (2018) 978e987
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 are
means ± standard deviation of triplicate measurements.
harbor the genes that encode shiga toxin, enterotoxin, virulent
factor, and adhesion toxin (Quiro
s and Muniesa, 2017). Meanwhile,
high throughput sequencing technology was used to evaluate the
impact of combined treatment on the diversity of soil indigenous
and lettuce endophytic bacterial communities. It was found that
the 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 roots
and leaves. Additionally, the combined treatment obtained the
highest microbial diversity in soil and lettuce, following by biochar
amendment, polyvalent phage inoculation, and controls. Previous
research has reported the role of biochar in improving hydrother-
mal ventilation and nutrient cycling in soil. Similarly, this work also
demonstrated its use as an environmentally-friendly soil condi-
tioner (Rajapaksha et al., 2015; Vithanage et al., 2014). When only
biochar was applied, the soil bacterial diversity was enhanced
significantly. 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, the
proportions of K-12 and PAO1 in soil and lettuce both decreased
dramatically to 2.4% and 2.9% compared to 4.0% and 8.8% in con-
trols. In contrast to the biochar amendment, sole inoculation of
polyvalent phage resulted in reduced diversities of soil and lettuce
endophytic bacterial communities compared to controls (p < 0.05).
This was likely caused by the polyvalent feature of the phage used
here. Given that it could infect both K-12 and PAO1 and use them as
hosts, it was quite possible for the phage to keep evolving and
develop a broader range of hosts in the soil (Amarillas et al., 2013;
Hsieh et al., 2011; Mahmoud et al., 2018). As the abundance of
microorganisms in the soil environment was much lower than that
985
occurring in the medium, it would be of great advantage to examine
a 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 biochar
and polyvalent phage were applied simultaneously, the diversity of
bacterial communities was restored compared to controls
(p < 0.05). In addition, a higher diversity of beneficial bacteria was
found 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 of
biochar 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 the
combined treatment (Table S5). Therefore, it is safe to conclude that
the technique combining biochar and polyvalent phage not only
significantly decreased the environmental risk by dissipating ARPB/
ARGs, but also restored the biological functioning of microbial
communities in the soil-lettuce system. Meanwhile, considering
the complex types of contaminated soil that exist, it is essential to
carry out further research to apply the combined technique to the
remediation of ARG/ARPB contaminated soils.
4. Conclusions
A novel biotechnology combining biochar and polyvalent phage
was developed in this study to stimulate the dissipation of
antibiotic-resistant pathogens in a soil-lettuce system. After 63
days of incubation, the dissemination of E. coli K-12 (tetR) and
986 M. Ye et al. / Environmental Pollution 241 (2018) 978e987
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-throughput
sequencing 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.
P. aeruginosa PAO1 (ampCR þ fosAR) was significantly impeded from
soil to lettuce tissues. In addition, ARG abundance in the soil-lettuce
system declined clearly. High throughput sequencing technology
analysis indicated the positive effect of the combined treatment on
the restoration of the microbial communities in the soil system.
This work provides novel information regarding the targeted
inactivation of multiple ARPB and ARGs, therein controlling their
dissemination risks in soil-vegetable-human systems.
Acknowledgement
This work was financially supported by the Environmental
Protection Research Project in Jiangsu Provincial Environmental
Department (2017005), the Jiangsu Agricultural Science and Tech-
nology Innovation Fund (CX(17)3047), the Scientific Apparatus
Research and Development Program of Chinese Academy of Sci-
ences (Young Talents: YJKYYQ20170057), the Jiangsu Municipal
Natural Science Foundation (BK20141050), the Leading Project of
the 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 at
https://doi.org/10.1016/j.envpol.2018.04.070.
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