Environment International 183 (2024) 108431

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Environment International
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Full length article

Long-term application of organic fertilizer prompting the dispersal of

antibiotic resistance genes and their health risks in the soil plastisphere
Da Lin a, b, Jia-Yang Xu a, b, Lu Wang a, c, Shuai Du a, c, *, Dong Zhu a, c, *
a
Key Laboratory of Urban Environment and Health, Ningbo Urban Environment Observation and Research Station, Institute of Urban Environment, Chinese Academy of
Sciences, Xiamen 361021, China
b
University of Chinese Academy of Sciences, 19A Yuquan Road, Beijing 100049, China
c
Zhejiang Key Laboratory of Urban Environmental Processes and Pollution Control, CAS Haixi Industrial Technology Innovation Center in Beilun, Ningbo 315830, China

A R T I C L E I N F O A B S T R A C T

Handling Editor: Dr. Hefa Cheng Microplastic (MP) pollution is a rapidly growing global environmental concern that has led to the emergence of a
new environmental compartment, the plastisphere, which is a hotspot for the accumulation of antibiotic resis­
Keywords: tance genes (ARGs) and human bacterial pathogens (HBPs). However, studies on the effects of long-term organic
Microplastics fertilizer application on the dispersal of ARGs and virulence factor genes (VFGs) in the plastisphere of farmland
Long-term organic fertilizer application
soil have been limited. Here, we performed a field culture experiment by burying nylon bags filled with MPs in
Paddy soil
paddy soil that had been treated with different fertilizers for over 30 years to explore the changes of ARGs and
Antibiotic resistome
Virulence factors VFGs in soil plastisphere. Our results show that the soil plastisphere amplified the ARG and VFG pollution caused
Human bacterial pathogens by organic fertilization by 1.5 and 1.4 times, respectively. And it also led to a 2.7-fold increase in the risk of
horizontal gene transfer. Meanwhile, the plastisphere tended to promote deterministic process in the community
assembly of HBPs, with an increase of 1.4 times. Network analysis found a significant correlation between ARGs,
VFGs, and bacteria in plastisphere. Correlation analysis highlight the important role of mobile genetic elements
(MGEs) and bacterial communities in shaping the abundance of ARGs and VFGs, respectively. Our findings
provide new insights into the health risk associated with the soil plastisphere due ARGs and VFGs derived from
organic fertilizers.

1. Introduction human activities, an abundance of VFGs primarily associated with

Antibiotic resistance genes (ARGs) in the environment are an
emerging pollutant that have been studied extensively (Banerjee and
van der Heijden, 2023; Singh et al., 2023). Annual deaths due to ARG
pollution in 2019 was 1.27 million, and this is projected to increase to 10
million by 2050 (Blackman et al., 2022). The presence of ARGs in soil is
especially concerning because soil is widely recognized as both the
reservoir and sink for ARGs (Bahram et al., 2018). Most studies have
primarily concentrated on the overall abundance of ARGs, often over­
looking another crucial factor that profoundly influences human, animal
and ecosystem health: virulence factors. Virulence factor genes (VFGs)
are genetic elements associated with the pathogenicity of bacteria that
cause infections and diseases in organisms (Diard and Hardt, 2017).
Currently, a wide variety of VFGs have been identified in different types
of soil. For instance, in the Arctic permafrost region untouched by
adhesion and invasion factors has been discovered (Kim et al., 2022).
Furthermore, agricultural soils that receive organic fertilizer application
have shown a higher abundance of VFGs (Zhu et al., 2022a). Human
bacterial pathogens (HBPs) harboring ARGs and VFGs in soil can
potentially be transmitted to humans through the food chain (Mussa
et al., 2022). These antibiotic-resistant HBPs can potentially survive
clinical treatments, presenting a substantial threat to human health
(Zhang et al., 2022). However, there is still a lack of research that fo­
cuses on both ARGs and VFGs.
Microplastic (MP) pollution poses a significant issue that has
garnered the attention of the United Nations Environment Program
(UNEP) (UNEP, 2018). MPs are minuscule (<5 mm) plastic particles that
have the potential to accumulate in soil, particularly in farmland soil.
They may originate from the utilization of organic fertilizer, leftover
plastic films from mulching, atmospheric deposition, and other

* Corresponding authors at: Key Laboratory of Urban Environment and Health, Ningbo Urban Environment Observation and Research Station, Institute of Urban
Environment, Chinese Academy of Sciences, Xiamen 361021, China.
E-mail addresses: sdu@iue.ac.cn (S. Du), dzhu@iue.ac.cn (D. Zhu).

https://doi.org/10.1016/j.envint.2024.108431
Received 18 September 2023; Received in revised form 5 January 2024; Accepted 5 January 2024
Available online 6 January 2024
0160-4120/© 2024 The Author(s). Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-
nc-nd/4.0/).
D. Lin et al.
pathways (Wang et al., 2022a). The amount of plastic released into the
terrestrial environment is estimated to be 4–23 times higher than that
released into the marine environment (Horton et al., 2017). Due to their
low biodegradability, considerable quantities of MPs persist in the soil
environment (Niu et al., 2022). Furthermore, microbial communities
can attach themselves to the surfaces of these particles, leading to the
formation of biofilm communities that are referred to as the plastisphere
(Amaral-Zettler et al., 2020). Because of their persistence, MPs may
create a hotspot for the acquisition and dissemination of ARGs and VFGs
in the plastisphere (Zhu et al., 2022b). Although there is growing
awareness of ARGs and VFGs in the environment, the relationship be­
tween these pollutants and the soil plastisphere remains unclear, espe­
cially in real farmland soil. Further investigation is necessary to fully
comprehend how ARGs and VFGs are dispersed, as well as the potential
risks associated with their presence in the plastisphere.
Organic fertilizers play a significant role in the proliferation of ARGs
and VFGs within the soil ecosystem (Zheng et al., 2022). Furthermore,
the composition of bacterial communities and MGE abundance are also
important factors affecting the abundance of ARGs and VFGs in soil
(Wang et al., 2022b). The selective pressure induced by soil pollutants,
such as antibiotics and heavy metals, as well as alterations in soil
properties such as pH, organic matter (OM), and total nitrogen (TN), can
regulate the bacterial community and MGEs in soil, which might sub­
sequently result in the changes of ARGs and VFGs (Wu et al., 2023a).
However, the effects of organic fertilizer on ARGs and VFGs in the soil
plastisphere and the impact of key factors on ARGs and VFGs in the soil
plastisphere are largely unknown. Pollutants introduced by organic
fertilizers in soil, including antibiotics, ARGs, HBPs, and VFGs, can
accumulate on MPs, creating a hotspot for the transfer of ARGs and VFGs
among different bacterial communities (Xu et al., 2020). Consequently,
it is likely that MPs amplify the health risks associated with ARGs and
VFGs that arise from the use of organic fertilizer. However, little is
known about this possibility. Paddy soil is prevalent globally, and its
quality and safety are intricately related to human health (Ali et al.,
2020). The long-term use of artificial farming practices inevitably leads
to the introduction of organic fertilizers and their associated ARGs and
VFGs into paddy soil, thereby turning it into a reservoir and sink for
pollution accumulation (Wu et al., 2023a). Meanwhile, paddy soil is a
complex agricultural soil that forms a water-soil interface system, in
which ARGs and VFGs on MPs can easily be disseminated and dispersed
through water flow (Zhao et al., 2021). As a result, the pollution of MP,
ARGs and VFG in paddy soil will bring harm and threat to the soil
environment. Studying such soils is of the utmost importance and
urgency.
Here, we employed a metagenomic approach to study the effects of
long-term fertilizer application on ARGs and VFGs of the plastisphere in
paddy soil. The objectives of our study were (1) to investigate the impact
of different fertilizer types on ARGs and VFGs in paddy soil; (2) to
compare the risks of ARGs and VFGs associated with soil, glass beads
(used as positive controls for MPs), and MPs in paddy soil receiving long-
term fertilizer application; (3) to reveal the change of potential human
bacterial pathogens in the soil plastisphere; (4) to assess the potential
health risks of ARGs and VFGs in the soil plastisphere using co-contig
and network analysis; and (5) to clarify the mechanisms underlying
the changes of health risks of ARGs and VFGs associated with soil
plastisphere. This study provides essential insights into the dissemina­
tion and risk of ARGs and VFGs in the soil plastisphere, thereby shedding
light on the potential consequences of MPs in the soil ecosystem.
2. Materials and methods
2.1. Field experiment setup
The experiment was conducted at the Zhejiang Academy of Agri­
cultural Sciences Experimental Station, which is located in Hangzhou,
China (120◦ 25′01′′ E, 30◦ 26′04′′ N). The experiment station has a
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Environment International 183 (2024) 108431
planting history of over 30 years, and its soil is classified as paddy soil
(loamy sand). Our study included the following treatments: a control
(CK, no fertilization), 750 kg/ha inorganic fertilizer (IF), 22.5 t/ha
organic fertilizer (OF), and a mixture of 750 kg/ha inorganic and 22.5 t/
ha organic fertilizer (MF, a commonly used soil fertilization method in
local area). The inorganic fertilizer consisted of 50 % urea, 25 % su­
perphosphate, and 25 % potassium chloride, while the organic fertilizer
consisted of swine manure. This experimental field practices biannual
crop rotation and applies either organic or chemical fertilizer as a base
fertilizer prior to each sowing. Each treatment was conducted in tripli­
cate, with an area of 300 m2. The soil has the following properties: pH
6.6; cation exchange capacity, 13.7 cmol/kg; organic matter, 28.7 g/kg;
total N, 1.67 g/kg; total P, 2.53 g/kg; total K, 21.8 g/kg.
After rice planting and flooding, we placed 30-μm glass beads and
polyethylene MPs into nylon mesh bags and buried them in the different
soil treatments. More details on MPs culture are described in our pre­
vious paper (Zhu et al., 2022b). The 30-μm glass bead treatment was
included as a positive control to exclude the influence that 30-μm par­
ticles and the mesh bag may have on the bacterial community (Xiao
et al., 2023). Each treatment was replicated three times. After three
months of cultivation, samples of soil, glass beads, and MPs from each
treatment were collected as the rice approached maturity, to conduct
subsequent analysis.
2.2. Determination of soil properties
Soil pH, OM, TC, and total nitrogen (TN) were measured using
conventional methods (Lu, 1999). Soil pH was measured in a mixture of
soil and water at a soil-to-water ratio of 1:2.5 (w/w). OM was estimated
using the K2Cr2O7 oxidation–reduction colorimetric method. TC and TN
were determined using the Kjeldahl and molybdenum antimony color­
imetry methods, respectively.
Heavy metals (As, Cd, Cu, and Zn) were analyzed by first digesting
soil samples in a HCl-HNO3-HF solution (2:3:1, v/v/v) and carrying out
the digestion in a MARS6 microwave system (CEM, USA). The concen­
trations of As, Cd, Cu, and Zn were then measured by inductively
coupled plasma mass spectrometry (ICP-MS) using an Agilent 7500a
system (USA).
We measured the levels of 23 antibiotics belonging to three classes
(sulfonamides, fluoroquinolones, and tetracyclines) that are often
detected in arable soil (Lin et al., 2023). Details of the antibiotics
measured in this study are found in the supplementary information. The
concentrations of antibiotics were measured by liquid chromatography
coupled with tandem mass spectrometry (LC-MS/MS) using a Dionex
instrument (USA) (Tang et al., 2020). The LC-MS/MS conditions and
other details of the antibiotic measurement procedures are provided in
the supplementary information.
2.3. Metagenomic sequencing and bacterial community analysis
DNA from the soil, glass beads, and PE plastispheres was extracted
using a Fast DNA spin kit (MP Biomedicals, Cleveland, USA), according
to the manufacturer’s instructions. The quality and concentration of the
extracted DNA were verified using 1 % agarose gel electrophoresis and a
Nanodrop 1000 instrument, respectively. The DNA was sequenced using
an Illumina Hiseq 2000 platform (Sequencing Depth: 6G) operated by
Majorbio Biotechnology Co. Ltd (Shanghai, China). The software fastp
was employed to remove low-quality reads, i.e., those with Q scores ≤
20, lengths < 50, and those containing N base (Chen et al., 2018). A total
of 419.5 Gb of “clean” data were generated, which were then assembled
using MEGAHIT (Li et al., 2015) (Table S1). Sequences with overlap
lengths (contigs) of > 300 bp were selected for further analysis. Open
reading frames (ORFs) were predicted by Prodigal software (Korandla
et al., 2020), and the non-redundant gene catalog was established by
CD-HIT (Fu et al., 2012). The raw metagenomic sequencing data were
deposited in the National Center for Biotechnology Information
D. Lin et al.
Sequence Read Archive under the Bioproject accession number
PRJNA988415.
Bacterial annotation and taxonomic information were obtained by
comparing representative sequences in the redundant gene set to those
present in the NCBI NR database (E-value ≤ 1 × 10− 5). Bacteria were
classified as potential HBPs by referring to a compilation of potential
HBPs from previous studies (Zhu et al., 2022).
2.4. Characterization of ARGs, VFGs, and MGEs
Alterations in the ARGs, MGEs, and VFGs were analyzed using local
ARG-OAP (version 3.2) based on the structured ARG (SARG) (Yin et al.,
2023), MGE (Pärnänen et al., 2018), and VFDB (Liu et al., 2022) data­
bases (E-value ≤ 10− 7, identity ≥ 80 % and hit length ≥ 75 %),
respectively. The SARG database consists of 30 different types and 2744
subtypes of ARGs; the MGE database includes annotations for 278
different gene names and over 2000 unique sequences; and the VFDB
database contains 14 different types of VFGs that are encoded by over
2000 genes. To compare the relative abundances of ARGs, MGEs, and
VFGs across different samples, their quantities were normalized relative
to the total cell abundance in each sample (copies/cell). ARGs and MGEs
were scored as co-contig if they were located on the same contigs (Song
et al., 2022a).
We identified high-risk ARGs according to the list of high-risk ARGs
constructed by Zhang et al., 2021. These ARGs are divided into three
categories: WHO-listed ARGs (i.e., known high-risk ARG families that
are recognized by the WHO and literature review), Rank I ARG families
(i.e., those enriched in human-related environments, possess gene
mobility, and are present in ESKAPE pathogens), and Rank II ARG
families (i.e., those that meet the first two criteria of Rank I ARG families
but not the third). This analysis allows for a comprehensive under­
standing the level of risk associated with ARGs in the environment.
2.5. Statistical analysis
Principal coordinate analysis (PCoA) cluster analysis based on the
Bray-Curtis distance and similarities analysis (ANOSIM) were employed
to evaluate differences in ARGs, VFGs, MGEs, and bacterial community
among soil, glass beads and MPs under different treatments, using the
“vegan” package in R (4.0.5). Bacterial diversity (Shannon), the visu­
alized of bar graph, bubble graph, and heatmaps were generated using
the packages “vegan” and “ggplot2”. The relationships between ARGs,
VFGs, MGEs, and bacterial communities were analyzed by linear
regression using the “ggplot2” package (Wen et al., 2023). Linear
discriminant analysis effect size (LEfSe) was used to determine whether
ARGs and VFGs in MPs were sensitive to different types of fertilizer. The
assembly mechanism of the bacterial community was analyzed using the
neutral community model (Sloan et al., 2006) while the extent of
deterministic processes contributing to the bacterial assembly was
evaluated using the R code from Burns et al., 2016. The relationships
between bacteria, ARGs, VFGs, and MGEs in soil, glass beads, and MPs
were evaluated by co-occurrence analysis of 100 bacterial genera, 100
ARG subtypes, 100 VFGs, and 50 MGE subtypes that were selected based
their relatively high abundance in each of the three treatments. Co-
occurrence networks of bacterial taxa, ARGs, VFGs, and MGEs (Spear­
man’s R2 > 0.7, P < 0.05) were analyzed using the “WGCNA” package
(Wen et al., 2023). The ecological networks, calculation of topological
parameters and modular analysis were performed in Gephi interactive
platform. The structural equation model (SEM) using the AMOS 28 (IBM
SPSS Inc., USA) and random forest analysis were used to uncover po­
tential factors affecting ARGs and VFGs. Statistical significance was
evaluated using one-way analysis of variance (ANOVA) in SPSS V26
software (IBM Inc., Chicago, USA), with P < 0.05 was considered sta­
tistically significant.
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Environment International 183 (2024) 108431
3. Results
3.1. Effects of long-term fertilization on the ARGs and VFGs
Analysis of the changes in soil properties during our experiment
showed that the MF and OF treatments all had significantly higher
contents of nutrients (OM, TN, and TP), heavy metals (Cd, Cu, and Zn),
and antibiotics compared to the CK (P < 0.05, Table S2-S4 and Fig. S1).
The soil pH had an average value of 7.4.
In total, 26 ARG types and 656 ARG subtypes were detected in all
samples. The dominant ARG types were those involved in multidrug
(23.7 %), bacitracin (15.8 %), and rifamycin (11.1 %) resistance
(Fig. 1A). ARGs were more abundant in the MF and OF treatments
compared to the other treatments (P < 0.05). Notably, ARGs related to
sulfonamide resistance were more abundant in the MF and OF treat­
ments, and these were particularly enriched in the plastisphere (3.8-fold
higher in OF treatments of plastisphere compared to OF treatments of
soil) (P < 0.05, Fig. 1A).
A total of 2261 unique VFGs were identified in the all samples. VFGs
were classified into 14 categories, and those related to adherence (29.1
%), immune modulation (20.3 %), nutritional/metabolic factor (14.2
%), and effector delivery system (10.8 %) were the most prevalent
(Fig. 1B). The VFGs of the OF group in the soil plastisphere (4.1 copies/
cell) was significantly more abundant than that of the control (3.8
copies/cell) (P < 0.05). Additionally, the VFGs were markedly more
abundant in the plastisphere than in soils (from 2.8 copies/cell to 3.0
copies/cell) or on glass beads (from 2.9 copies/cell to 3.4 copies/cell) (P
< 0.05, Fig. 1B).
The higher abundance of ARGs and VFGs in plastisphere may be
linked to an intensified capacity for horizontal gene transfer (HGT).
Similarly, we identified other MGEs, including transposase, insertion
sequences (IS), qacEdelta, integrase, tniB, and istB (Fig. S2). The total
abundance of MGEs in the MF and OF treatments were significantly
higher than those in the CK treatment (P < 0.05). In particular, MGEs
were enriched in the soil plastisphere, where MGE abundance in the OF
treatment was 2.6- and 1.5-fold higher than in the OF treatment of soil
and glass beads, respectively (P < 0.05, Fig. S2). The results of PCoA
revealed significantly different profiles of ARGs, VFGs, and MGEs in the
soil plastisphere compared to those in both soils and glass beads
(ANOSIM, P < 0.05) (Table S5, Fig. 1C and 1D and Fig. S3).
3.2. Risk of ARGs and VFGs in soil plastisphere
We observed that Rank I ARGs were most abundant in the MPs group
(P < 0.05, Fig. 2A). And the abundance of WHO-listed ARGs were
extremely low in the untreated group (P < 0.05). However, their levels
were very high in the organic fertilizer treatment (MF and OF), espe­
cially in the MPs group (P < 0.05, Fig. 2A). Specifically, the high
abundance of Rank I ARGs in the MPs group, as well as the greatly in­
crease of WHO-listed ARGs under organic fertilizer application, can be
attributed primarily to the elevated levels of bacA gene and change in
the sul1 gene within the MPs, respectively (P < 0.05, Fig. 2B).
The average abundance of bacA gene across all treatments with MPs
was 0.0245 copies/cell, which is significantly higher than 0.0189
copies/cell in soil and 0.0224 copies/cell in glass beads (P < 0.05).
Within the MPs, the abundance of sul1 gene in the CK group was
0.000712 copies/cell, while that in the OF group was 0.0327 copies/
cell, which represents an approximately 45.9-fold difference (P < 0.05,
Fig. 2B). Lefse analysis was conducted to examine the features of the top
50 ARG subtypes in the MPs across different treatments, and it found the
sul1 gene specifically in OF treatment and had the highest linear
discriminant analysis (LDA) score, suggesting that it is extremely
involved in the ARG pollution caused by organic fertilizer in the soil
plastisphere (Fig. S4). A similar Lefse analysis of the top 50 VFGs in the
plastisphere also demonstrated that the biomarker genes like groEL2,
panD, mpa, glnA1, and pafA were enriched in the OF treatment (P < 0.05,
D. Lin et al. Environment International 183 (2024) 108431

Fig. 1. Change of ARGs and VFGs in soil, glass bead and MPs amended with different fertilizers. The abundance (left) and fold change (right) of (A) ARGs and (B)
VFGs. The fold change represents the differences in various treatments between the glass bead and MPs groups compared to the soil group. Different lowercase letters
show significant differences among the treatments (P < 0.05). Principal coordinate analysis (PCoA) cluster analysis based on Bray − Curtis distances show the
patterns of (C) ARGs and (D) VFGs.

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D. Lin et al. Environment International 183 (2024) 108431

Fig. 2. The change of high-risk ARGs and VFGs with high abundance in soil, glass bead and MPs amended with different fertilizers. (A) The total abundance of high-
risk ARGs. The bubble graph illustrates the changes in (B) high-risk ARGs at three levels and (C) VFGs with high abundance among different treatments. Different
lowercase letters show significant differences among the treatments (P < 0.05).

Fig. 3. The co-occurrence patterns of ARGs and MGEs on the contigs in the MPs.

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D. Lin et al. Environment International 183 (2024) 108431

Fig. S5), i.e., abundance of these genes were 3.6, 9.8, 3.7, 3.5, and 3.8
times higher than their abundance in the OF treatment of soil, respec­
tively (P < 0.05, Fig. 2C).
The abundance of ARGs, VFGs, and MGEs were positively correlated,
particularly in the soil plastisphere (P < 0.05). In particular, MGEs were
strongly correlated to both ARGs (R = 0.98, P < 0.01) and VFGs (R =
0.73, P < 0.01) in the MPs. And in soil, MGEs were correlated to ARGs
and VFGs at R values of 0.89 (P < 0.01) and 0.43 (P < 0.05), respec­
tively; and in glass beads, MGEs were correlated to ARGs and VFGs at R
values of 0.94 (P < 0.01) and 0.62 (P < 0.01), respectively (Fig. S6).
Fig. 3 shows 16 representative co-occurrence patterns between ARGs
and MGEs in the soil plastisphere, which further emphasizes the direct
correlation between ARGs and MGEs. Additionally, all these represen­
tative co-occurrence patterns were observed exclusively in the organic
fertilizer application groups (MF and OF), indicating that the application
of organic fertilizer led to an increased frequency of co-occurrence be­
tween ARGs and MGEs. Notably, it was observed that the sul1 gene and
MGE frequently co-occurred eight times.
3.3. Potential human bacterial pathogens in the soil plastisphere
The bacterial community differed significantly between soil, glass

Fig. 4. Bacterial community and HBPs composition in soil, glass bead and MPs amended with different fertilizers. (A) Composition of bacteria in different treatments.
(B) Abundance of potential HBPs at phylum level. Different lowercase letters show significant differences among the treatments (P < 0.05). (C) Inner and outer
heatmaps indicate relative abundance and fold change of the top 40 HBPs among different treatments, respectively. The fold change represents the differences in
various treatments between the glass bead and MPs groups compared to the soil group.

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D. Lin et al.
beads, and MPs, with those in soil and MPs differing to a greater extent
(ANOSIM, R = 0.875, P = 0.001) compared to those in glass beads and
soils (ANOSIM, R = 0.613, P = 0.001) (Table S5 and Fig. S7). Proteo­
bacteria (34.7 %) and Actinobacteria (30.2 %) were the dominant bac­
terial phyla in MPs. Furthermore, the proportion of Actinobacteria in the
MPs was markedly higher than those in soil (12.2 %) and glass beads
(15.9 %) (P < 0.01, Fig. 4A). The Shannon index of bacteria in the MPs
was significantly lower than those in soils and glass beads (P < 0.05,
Fig. S8).
The application of organic fertilizer led to a substantial increase in
the abundance of potential HBPs in soil, glass beads, and MPs. In
particular, we observed a noticeable enrichment of potential HBPs in
MPs, which is consistent with the trend observed for ARGs and VFGs (P
< 0.05, Fig. 4B). Analysis of the 40 most abundant HBPs revealed that
the five most abundant species were Saccharomonospora viridis (16.1 %),
Cystobacter fuscus (3.6 %), Streptococcus pneumoniae (3.1 %), Bur­
kholderia pseudomallei (2.6 %), and Bacillus cereus (2.5 %). OF treatment
in MPs induced the increase of most HBPs, particularly Rhodococcus
fascians, Mycobacterium gordonae, Amycolatopsis orientalis, Mycobacte­
rium marinum and Mycobacterium avium, which were 16.5, 6.6, 5.3, 4.8,
and 4.3 times more abundant than OF treatment in soil, respectively (P
< 0.05, Fig. 4C). The neutral community model base on bacterial
community revealed that more bacteria (74.0 %) and HBPs (71.0 %) in
MPs were affected by deterministic processes compared to those in soils
(bacteria: 49.4 % and HBPs: 51.6 %) and glass beads (bacteria: 47.0 %
and HBPs: 49.4 %). This suggests that deterministic processes may play a
more prominent role in assembling the bacterial community in the MPs
(Fig. S9 and S10).
3.4. Co-occurrence patterns among ARGs, VFGs, MGEs, and bacterial
taxa
The results of network analysis show that bacterial taxa in the MPs
exhibited stronger associations with ARGs, VFGs, and MGEs than those
in soil and glass beads; moreover, they tend to have a larger node
number, edge number, average degree, and modularity (Fig. 5A). We
found four clusters of ARGs, VFGs, and MGEs strongly co-existing with
bacteria in the networks of soil, glass beads, and MPs. Within module 1
of the MP network, we observed significant positive correlations be­
tween the abundance of ARGs, VFGs, and bacteria (P < 0.01). This result
suggests that bacteria present in module 1 can potentially contribute to
the increased pollution of ARGs and VFGs in the MPs. Further analysis of
the bacterial composition in module 1 showed that it predominantly
consists of Actinobacteria and Proteobacteria (Fig. S11). The abundance
of these taxa increased following MF and OF application (P < 0.01). In
addition, we observed that g_unclassified_c_Actinobacteria and Nocar­
dioides were the most dominant genera of Actinobacteria while
g_unclassified_c_Deltaproteobacteria and Methylomonas were the most
dominant genera of Proteobacteria in module 1 (Fig. S12).
3.5. Effect of different factors on ARGs and VFGs
SEM analysis was performed to examine the effect of fertilizer,
ecological niche (represented by three different habitats: soil, glass
beads, and MPs), soil properties (including OM, TN, TP, pH, heavy
metal, and antibiotic), bacterial community (bacterial abundance and
bacterial diversity), and MGEs on ARGs and VFGs. MGEs clearly (λ =
0.838, P < 0.01) had the most significant direct impact on ARGs, while
both bacterial abundance (λ = − 0.427, P < 0.01) and bacterial di­
versity (λ = − 0.262, P < 0.01) were negatively correlated to VFGs
(Fig. 6A). Soil properties also had a direct relationship with MGEs (λ =
0.821, P < 0.01). Analysis of the standardized direct and indirect effects
on ARGs and VFGs show that long term fertilization and ecological niche
were positively correlated with ARGs and VFGs (Fig. 6B). Moreover, soil
property has a stronger impact on the ARGs than VFGs, is consistent with
Random Forest analysis (Fig. 6B, 6C and 6D). Further investigation
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Environment International 183 (2024) 108431
determined that the effect of soil properties on ARGs can be attributed
primarily to the influences of antibiotics and heavy metals (acting as
selective pressures), as well as those of TN and OM, which are indicators
of soil physicochemical (Wang et al., 2024). Based on random forest
ranking, MGEs were the most important predictor for ARGs, while the
bacterial community (bacterial abundance and bacterial diversity) were
the most significant predictor for VFGs (Fig. 6C and 6D).
4. Discussion
4.1. Soil plastisphere amplified the risk of ARGs and VFGs
The results of our study reveal that compared to soils and glass beads,
ARGs and VFGs were most enriched in the MPs. Similar findings have
been reported in other studies (Song et al., 2022b; Zhu et al., 2022b).
Furthermore, the enrichment of ARG and VFG in MPs were enhanced by
the application of organic fertilizer. Analysis of the abundance of high-
risk ARGs and VFGs in the plastisphere revealed the enrichment of bacA
in the soil plastisphere, which is concerning because bacA is classified as
a rank I high-risk ARG. This finding aligns with previous reports of a
high prevalence of the bacA gene in MPs (Song et al., 2022b). Similarly,
compared to soil and glass beads, the sul1 gene was most enriched in the
soil plastisphere due to organic fertilizer application. The sul1 gene is
classified as a WHO-listed ARG that is commonly found in the envi­
ronment, particularly in areas prone to anthropogenic contamination
(Zhang et al., 2021). This finding suggests that ARGs introduced through
human activities tend to accumulate in the plastisphere, particularly in
soils that have received long-term manure application. The application
of organic fertilizer also led to an increased abundance of groEL2 in the
soil plastisphere. This VFG plays a crucial role in regulating macrophage
phagocytosis and is frequently encountered in HBPs like Mycobacterium
tuberculosis (Vinod et al., 2021). The results of linear regression analysis
reveal a higher risk of HGT in the plastisphere compared to those in soil
and glass beads, which is consistent with the results of a previous study
(Yu et al., 2023). Within MPs, we frequently observed ARG and MGE co-
located in the same contig, especially for the sul1 gene, which suggests a
higher occurrence of horizontal transfer of ARGs in the soil plastisphere
and may explain why sul1 was enriched in the soil plastisphere under
organic fertilizer application.
4.2. Soil plastisphere enhanced the correlation among ARGs, VFGs, and
bacteria
The soil plastisphere serves as a unique habitat for environmental
microorganisms due to its capacity to support the colonization of a
diverse array of bacteria, thereby establishing a miniature ecosystem
(niches for microbiota) (Li et al., 2021). The processes acting on bac­
terial communities are more deterministic on MPs than in soil or glass
beads. Therefore, MPs offer a unique ecological niche with a selective
preference for the growth of specific bacterial groups (e.g., Actino­
bacteria), and it has the potential to create favorable conditions for the
growth and proliferation of HBPs (Zhu et al., 2023). Consequently, MPs
are associated with low levels of diversity and abundance of soil bacteria
while promoting the proliferation of HBPs. In addition, the soil plasti­
sphere contributes to the enrichment of bacterial taxa such as those in
module 1 of the co-occurrence network. These bacterial taxa tend to co-
occur with ARGs, VFGs, and MGEs, and they consist primarily of Acti­
nobacteria and Proteobacteria, which have previously been identified as
the primary host bacteria for ARGs and VFGs (Kim et al., 2022; Qian
et al., 2021; Zheng et al., 2022). For example, Actinomycetes is the main
group of antibiotic-producing bacteria. For the purpose of self-
protection, genes for antibiotic synthesis are often located in the same
gene cluster as ARGs and their regulatory elements. Therefore, bacteria
such as Actinomycetes can potentially serve as a source of ARGs (Jiang
et al., 2017; Thaker et al., 2013). Alternatively, Proteobacteria includes
numerous known pathogens (e.g., Escherichia coli, Salmonella, Vibrio
D. Lin et al. Environment International 183 (2024) 108431

Fig. 5. The relationship among bacteria, ARGs, VFGs and MGEs. (A) Network analysis revealing bacteria, ARGs, VFG and MGEs co-occurrence pattern in soil, glass
bead and MPs. The color of node was filled based on the bacteria, ARGs, VFGs, and MGEs group. (B) Network diagram with nodes colored according to each of the
four main clusters (modules 1–4). Line regression analysis between (C) total ARG abundance, (D) total VFG abundance and the relative abundance of bacteria in the
four main clusters.

8
D. Lin et al. Environment International 183 (2024) 108431

Fig. 6. The influence of various factors on ARGs and VFGs. (A) Structural equation models showing both the direct and indirect effects of long-term fertilizer,
ecological niche, soil property, bacterial abundance, bacterial diversity and MGEs on the abundance of ARGs and VFGs. The parameter of hypothetical models as
follows: χ2/DF = 2.229, P = 0.06, CFI = 0.949, RMSEA = 0.168. (B) Standardized direct and indirect effects derived from the structural equation models. Solid and
dashed line represent significant and non-significant relationships, respectively. Blue and black arrows represent positive and negative relationships, respectively.
Random Forest model revealing the importance of soil physicochemical (OM, TN, TP and pH), selective pressure (heavy metal and antibiotic), bacterial community
(bacterial abundance and bacterial diversity) and MGEs in predicting changes of (C) ARGs and (D) VFGs. The importance of predictors was estimated by calculating
the percentage increases in the mean squared error (MSE) of the variables, where higher MSE% values indicate a greater importance of the predictor. Significant
differences are denoted as *(P < 0.05) and **(P < 0.01). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of
this article.)

cholerae, Helicobacter pylori and Pseudomonas aeruginosa) that are
commonly found in soil and often acquire ARGs from other species such
as those in the group Actinomycetes (Klümper et al., 2015; Su et al.,
2015). Consequently, these two bacterial phyla are likely to play a
crucial role in the persistence of ARGs and VFGs in the environment.
Overall, our results suggest that certain bacteria selected by MPs may
also contribute to the enrichment of ARGs and VFGs in the soil
plastisphere.
4.3. Potential mechanisms of ARG and VFG pollution in soil plastisphere
We conducted SEM and random forest analysis to elucidate the un­
derlying mechanism involved in the enrichment of ARGs and VFGs in
the soil plastisphere. Our findings revealed that MGEs play a key role in
influencing ARGs, highlighting the role the soil plastisphere plays as a
hotspot for the transmission of ARGs among bacteria. These elements
can move both within or between DNA molecules, as well as transfer
between bacterial cells (Partridge et al., 2018). Therefore, these ele­
ments serve as an important means of acquiring and spreading ARGs.
The selective pressure exerted by antibiotics and heavy metals, which
are introduced through the application of organic fertilizer (Wu et al.,
2023), may indirectly influence ARGs by affecting MGEs. Moreover, we
observed that the bacterial community was the main factor affecting
VFGs, suggesting that high abundance of VFGs can be attributed to the
enrichment of HBPs and the collective behavior of bacterial populations
in the soil plastisphere. Bacteria can enhance their capacity to produce
and release virulence factors by collectively secreting pathogen effectors
(Ruiz-Bedoya et al., 2023). Based on our data and previous research, we
propose the following reasons contributing to the enrichment of ARGs
and VFGs in MPs compared to soil and glass beads under the application
of organic fertilizer. The inherent properties of MPs, such as electric
charge and hydrophobicity, can facilitate the accumulation of pollutants
(i.e heavy metals and antibiotics) introduced by organic fertilizer. This
has the potential to contribute to the selection and proliferation of
antibiotic-resistant bacteria by applying selective pressure (Wang et al.,
2024). In addition, bacteria can efficiently attach to the surface of MPs
to form biofilms that are supplied with nutrients from organic fertilizer
(Wu et al., 2019). The protective properties of biofilms enhance the
survival and activity of bacteria, thereby strengthening the interactions
among members of the biofilm communities and potentially promoting
the spread of ARGs and VFGs within these communities (Wu et al.,
2022). Furthermore, MPs often contain additives that can leach into the

9
D. Lin et al.
environment. These chemicals can also exert selective pressure resulting
in the dissemination of ARGs in the plastisphere, which enhances the
possibility of horizontal gene transfer (Xu et al., 2024). Therefore, the
plastisphere-mediated dispersal of ARGs and succession of virulence
factors within the soil ecosystem require further study.
5. Conclusion
In summary, we found that the soil plastisphere increased the
abundance of ARGs, VFGs, HBPs, and MGEs in paddy soil with long-term
application of organic fertilizer. This increase raises the risk of ARG
transfer within the soil ecosystem. Additionally, it triggers alterations in
the composition of bacterial community, thereby increasing the poten­
tial risk associated with HBPs. We also identified that module 1 pri­
marily consisted of Actinobacteria and Proteobacteria in the network of
soil plastisphere and exhibited a significant correlation with ARGs and
VFGs, as well as the mechanisms that affected ARGs and VFGs. In paddy
soil, the pollution of MPs along with their associated risks of ARGs and
VFGs were highly likely to spread through water flow and aquatic ani­
mal food webs. Overall, our study has comprehensively elucidated the
high dispersion and health risks of ARG and VFG pollution in soil plas­
tisphere based on read-based and contig-based analysis. These findings
effectively extend our knowledge on the potential hazards associated
with soil plastisphere under long-term human activities.
CRediT authorship contribution statement
Da Lin: Writing – original draft, Visualization, Methodology,
Investigation, Formal analysis, Data curation. Jia-Yang Xu: Visualiza­
tion, Formal analysis, Data curation. Lu Wang: Investigation. Shuai Du:
Writing – review & editing, Supervision, Conceptualization. Dong Zhu:
Writing – review & editing, Supervision, Resources, Project adminis­
tration, Funding acquisition, Conceptualization.
Declaration of competing interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Data availability
Data will be made available on request.
Acknowledgements
This work was funded by the National Natural Science Foundation of
China (22193062, 42222701, 42077216, 42207260 and 42207013),
Youth Innovation Promotion Association, Chinese Academy of Sciences
(2023321), Ningbo Yongjiang Talent Project (2022A-163-G) and
Ningbo S&T project (2021-DST-004). We appreciate Professor Yong-
Guan Zhu for his valuable comments and suggestions.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.envint.2024.108431.
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