Environmental Pollution 302 (2022) 119065

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

Metagenomic analysis of microbial community structure and distribution of

resistance genes in Daihai Lake, China☆
Caili Du a, b, Fang Yang a, *, Xiaoguang Li a, Haiqing Liao a, Zhonghong Li a, Jiayue Gao c,
Lieyu Zhang a
a
Chinese Research Academy of Environmental Science, Beijing 100012, China
b
College of Environmental Science and Engineering, Tongji University, Shanghai 200092, China
c
School of Space and Environment, Beihang University, Beijing 100191 China

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

Keywords: The emergence of resistance genes is a global phenomenon that poses a significant threat to both animals and
Metagenomic analysis humans. Lakes are important reservoirs of genes that confer resistant to antibiotics and metals. In this study, we
Resistance gene investigated the distribution and diversity of antibiotic resistance genes (ARGs) and metal resistance genes
Bacterial diversity
(MRGs) in the sediment of Daihai Lake using high-throughput sequencing and metagenomic analysis. The results
Sediment
indicated that all sampling sites had similar bacterial community structures, with Proteobacteria, Actinobacteria,
Daihai lake
Firmicutes, and Bacteroidetes being the most abundant. A total of 16 ARG types containing 111 ARG subtypes
were deposited in the sediment. Among the resistance genes to bacitracin, multidrug, macrolide-lincosamide-
streptogramin (MLS), tetracycline, beta-lactam, and sulfonamide were the dominant ARG types, accounting
for 89.9–94.3% of the total ARGs. Additionally, 15 MRG types consisting of 146 MRG subtypes were identified. In
all samples, MRGs of the same type presented resistance to Pb, Ni, Hg, W, Zn, Ag, Cr, Fe, As, Cu, and multimetals.
Overall, the distribution and diversity of antibiotic and metal resistance genes showed no significant differences
in the samples. Plasmids (91.03–91.82%) were the most dominant mobile genetic elements in the sediments of
Daihai Lake. Network analysis indicated that the target ARGs and MRGs were significantly positively correlated
with the microorganisms. Potential hosts for various ARGs and MRGs include Proteobacteria, Euryarchaeota,
Actinobacteria, Chloroflexi, and Bacteroidetes.

1. Introduction
Antibiotics have been extensively applied in various industries,
including medical treatment, veterinary, agriculture, and aquaculture,
which rising considered as emerging environmental contaminants (Hu
et al., 2013; Larsson and Flach, 2021; Song et al., 2019; Wang et al.,
2021). According to statistics, 9.27 × 104 t of antibiotics were consumed
in China, and 5.38 × 104 t of antibiotics were discharged into the
environment, particularly in rivers and soil via water (Qiao et al., 2018;
Zhang et al., 2015). Hence, overuse, misuse, and incorrect dosing of
these antibiotics leads to dispersal of antibiotic-resistant genes (ARGs)
and antibiotic-resistant bacteria in the environment (Wright, 2010).
Earlier studies reported that ARGs and antibiotic-resistant bacteria
(ARB) had been found in numerous habitats, including drinking water,
farmland soil, landfill leachate, and wastewater treatment plants
(Lekunberri et al., 2016; Wang et al., 2019b; Wu et al., 2014). ARGs can
be spread not only within the same species but also between different
bacterial strains via horizontal gene transfer (HGT) and mobile genetic
elements (MGEs), such as integrons (In), plasmids, insertion sequences
(IS), integrating conjugative elements (ICEs) and transposons (Tn)
(Gillings, 2017). More importantly, ARGs in the environment can be
transmitted and disseminated to human pathogens (Hawkey and Jones,
2009), posing a risk to humans (Pehrsson et al., 2016). In recent years,
there has been widespread concern about the impact of resistance genes
as emerging pollutants in the environment and for humans.
The lake ecosystem is regarded as an environmental reservoir for the
propagation, diffusion, and survival of resistance genes because it re­
ceives a variety of effluents, including wastewater treatment plant ef­
fluents, farm effluents, and effluents from urban residential areas and
hospitals (Lin et al., 2015; Ma et al., 2017; Yang et al., 2017). Studies

☆
This paper has been recommended for acceptance by Dr. Sarah Harmon.
* Corresponding author.
E-mail address: yang.fang@craes.org.cn (F. Yang).

https://doi.org/10.1016/j.envpol.2022.119065
Received 21 September 2021; Received in revised form 18 February 2022; Accepted 24 February 2022
Available online 25 February 2022
0269-7491/© 2022 Elsevier Ltd. All rights reserved.
C. Du et al.
have reported that these effluents could lead to the dispersal of such
genes in river ecosystems; the average relative abundance of resistance
genes from farm effluent was more than four times greater than from the
river (Rowe et al., 2016). Additional studies found that the most abun­
dant ARGs in the river catchment were tetracycline-resistant genes (tetC
and tetW) from farm effluents. The relative abundance of ARGs and
bacterial community components in sediment samples from receiving
water bodies is significantly influenced by effluents from wastewater
treatment plants (Chu et al., 2018). In addition to ARGs, metal resistance
genes (MRGs) have been discovered in several lakes worldwide (Stange
et al., 2019; Tong et al., 2020; Yang et al., 2019). The presence of MRGs
contributes to the spread and diffusion of ARGs (Salam, 2020). Bacteria
that harbor resistomes (MRGs and ARGs) are more likely to be con­
jugative and transferred among different bacterial strains (Pal et al.,
2015).
Most studies detected or quantified resistance genes in various en­
vironments using traditional PCR and qPCR techniques. Essentially,
these traditional techniques do not provide more information about
genetic diversity, except for the selected genes being amplified, which
are only suitable for certain species analyses. However, metagenomic
sequencing could be used to sequence and analyze the total DNA from
environmental samples, relying on cloning of random DNA fragments
into suitable vectors and not limited to specific primers, and the
throughput is improved by several orders of magnitude compared to the
qPCR techniques. This method might detect different species (bacteria,
fungi, and pathogens) and uncover information about new genes or new
species. In addition, the technology provides important information
about genomic features, functional genes, and the composition, func­
tion, and metabolic capacity of the bacterial community. Therefore, it is
an excellent method to understand bacterial communities and resistance
genes in environmental samples (Port et al., 2014).
Antibiotic resistance is an ancient and natural phenomenon that is
widespread in the natural environment (D’Costa et al., 2011). Lakes are
important carriers for the spread and propagation of resistant genes,
which provide services for humans and animals, such as safe drinking
water, farming water, and inland fisheries. Daihai Lake, a typical inland
lake in the semi-arid area of northwest China, has no outlet, and all
surface and underground runoff in the basin are discharged and
collected in the lake (Liang et al., 2014). It is alkaline and saline, with a
pH varying from 8 to 9. A coal-fired power plant, which began operation
in 2005, was built in the southwest region of the lake. The waste and
coal ash from coal-fired power plants may increase the pollutant input
(e.g., nutrients and heavy metals) to the lake. Therefore, the water
quality of the lake has deteriorated. Despite several measures to prevent
and control pollution in the basin, the lake remains severely eutrophic.
Because there is no outflow from the lake, external pollutants accumu­
late within the catchment. In addition, the rapid decline of the Daihai
water level will further increase the concentration of pollutants, has­
tening the trend of water quality deterioration and disrupting the
virtuous cycle of the lake water ecosystem. Moreover, there have been
no reports on the distribution, diversity, and co-occurrence patterns of
ARGs and MRGs in inland lakes. This study was the first to report the
distribution and components of resistomes in Daihai Lake based on
metagenomic sequencing. The aims of the present study were to: (1)
survey the structure or composition of the microbial community in
Daihai Lake; (2) characterize the distribution of ARGs and MGEs using
metagenomic analysis; and (3) identify the potential ARGs and MRGs
hosts through network analysis.
2. Materials and methods
2.1. Study area
Daihai Lake (40◦ 32′ to 40◦ 36′ N, 112◦ 37′ to 113◦ 46′ E) is located in
the eastern portion of Liangcheng City, Inner Mongolia, China, which is
a typical interior continental closed lake basin with a total area of 133.5
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Environmental Pollution 302 (2022) 119065
km2, an average water depth of 4 m (Sun et al., 2021). With an average
annual precipitation of 403.5 mm and average annual evaporation of
813 mm, precipitation is the primary recharge source for the basin.
Furthermore, precipitation in Daihai Lake is low and uneven during the
year. In addition, 22 seasonal rivers flow into the Daihai Basin for
replenishment (Huang and Chun, 2021). The primary rivers that enter
the lake are the Gongba in the west, Tiancheng in the south, and the
Muhua in the east (Huang and Chun, 2021). The majority of the rivers
surrounding the basin are dry for most of the year.
2.2. Field sampling
Based on the characteristics of the lake and inflowing rivers, eight
samples (S1-S8) were investigated in Daihai Lake (Fig. 1). Two (S1 and
S2), three (S3, S7, and S8), and three (S4, S5, S6) sampling sites were
located at the western, middle, and eastern regions of the lake, respec­
tively. The surficial sediment in each location was sampled from the top
5 cm layer using a Peterson sediment sampler in November 2020 (Jiang,
2012). For each sampling site, three sub-samples of surface sediments
were collected and mixed as one sample, stored in a 50-mL polyethylene
tube, and sent to the laboratory within 24 h on dry ice and stored at
− 80 ◦ C for further analysis.
2.3. DNA extraction and metagenomic sequencing
The total DNA from each sediment sample was extracted using the
FastDNA™ Spin Kit for Soil (MP Biomedicals, CA, USA), according to the
manufacturer’s instructions. The quality and integrity of all DNA sam­
ples were verified by 1% agarose gel electrophoresis.
All extracted DNA samples were sent to Beijing Nohe Zhiyuan Sci­
ence and Technology Co., Ltd. for Illumina shotgun high-throughput
sequencing using an Illumina Hiseq 2500 platform (2 × 150 bp).
2.4. Bioinformatic analysis
The original sequencing reads obtained from all sampling sites were
subjected to quality control using Trimmonatic (Bolger et al., 2014), and
the Kraken2 method was applied to analyze the microbial community
structure of all sampling sites (Wood et al., 2019). All clean reads were
matched and annotated as different types and subtypes of ARGs based on
the SARG database with USEARCH (ACCEL ≤0.5, e-value of 10− 5) (Yin
et al., 2018). Furthermore, all metagenomic sequencing was annotated
as MRGs based on the BacMet reference dataset with BLASTx (Pal et al.,
2014). Then, all clean data were also searched for MGEs against the IS
Finder database (https://isfinder.biotoul.fr/), INTEGRALL database
(http://integrall.bio.ua.pt/), ICEberg database (https://db-mml.sjtu.
edu.cn/ICEberg/ICE) and NCBI RefSeq databases by BLASTn. MGEs
including In, IS, and ICEs were only annotated when the e–value < 1e− 5,
the nucleotide identity ≥95%, and alignment length ≥50 bp. MGEs,
including plasmids and Tn, were identified when the sequence identity
was ≥95%, an alignment length ≥90 bp, and e-value < 1e− 5. The
number of ARGs and MRGs reads per million reads was used to stan­
dardize the average relative abundances of ARGs and MRGs.
2.5. Statistical analysis
Origin 2018 and R 4.1.0 were used for all data analysis and charting.
Network analysis based on Spearman’s rank correlation was used to
investigate the co-occurrence patterns among the resistomes, MGEs, and
microbial communities. Before correlation analysis, rare operational
taxonomic units (genus, relative abundance <0.2%) in each sample
were deleted. Correlations were constructed based on the abundance of
all ARGs, MRG subtypes in each sampling site, MGEs and, microbial
community by calculating Spearman’s rank correlation using the R
vegan, Igraph, and Hmisc packages. The study defined correlation co­
efficient values r ≥ 0.8 and P ≤ 0.05 between resistomes and
C. Du et al.
Fig. 1. Sampling site in Daihai Lake.
microorganisms as significant correlations. Network analysis was car­
ried out using Gephi software (version 0.9.2).
2.6. Sequence submission
The raw sequence reads of the metagenome used in this study have
been deposited in the Genome Sequence Archive (GSA) in National
Genomics Data Center, Beijing Institute of Genomics (China National
Center for Bioinformation), Chinese Academy of Sciences, under
accession number CRA005473 and are publicly accessible at https://big
d.big.ac.cn/gsa/browse/CRA005473.
3. Results
3.1. Bacterial community structure abundance in the sediment of Daihai
Lake
To investigate the microbial community diversity of all sediment
samples, an average of 44,931,128 effective sequences were generated.
Most metagenomic reads (95.4–98.6%) were taxonomically assigned to
bacteria in this study. The results showed that all sampling sites had
similar bacterial community structures. The phylum, Proteobacteria,
was the main bacterial community component, representing
76.4–82.6% of the total phyla in the given samples, followed by Acti­
nobacteria (7.03–8.18%). Additionally, some phyla dominated the mi­
crobial community structure in each sampling site, such as Firmicutes
and Bacteroidetes, accounting for 2.38–3.76% and 2.38–8.11%,
respectively (Fig. 2).
Fig. 2. Relative abundance of bacterial community structure at the phylum
level in the sediments of Daihai Lake.
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Environmental Pollution 302 (2022) 119065
3.2. Abundance of ARGs in the sediment of Daihai Lake
To understand the profile of ARGs in sediment samples from Daihai
Lake, we conducted a USEARCH analysis of all metagenome samples
against the SARG database. The ARGs were identified into 16 ARG types
via metagenomic analysis in S1 (14 types, 43 ppm), S2 (14 types, 36
ppm), S3 (13 types, 34 ppm), S4 (12 types, 38 ppm), S5 (16 types, 30
ppm), S6 (12 types, 36 ppm), S7 (13 types, 42 ppm), and S8 (12 types,
35 ppm). Among these types of ARG, 11 were shared at the selected
sampling sites. The predominant ARG types were resistance genes for
bacitracin, multidrug, macrolide-lincosamide-streptogramin (MLS),
tetracycline, beta-lactam, and sulfonamide in the given samples,
contributing approximately 89.9–94.3% of the total ARGs (Fig. 3).
Nonetheless, the relative abundances of the main ARGs varied across
different sampling sites: bacitracin was the dominant ARG in S1
(39.26%), S7 (42.27%), and S8 (45.37%); however, multidrug domi­
nated the other samples (S2, S3, S4, S5, and S6), accounting for
39.44–43.92% of the total relative abundance of ARGs (Fig. 3). Other
ARG types (resistance genes for chloramphenicol, trimethoprim, kasu­
gamycin, quinolone, rifamycin, aminoglycoside, and vancomycin) were
also detected; however, they were not as prevalent in the selected
sampling sites. Chloramphenicol resistance genes were only detected in
S5, with the lowest abundance at 0.02 ppm. Overall, the total relative
abundance of ARGs in Daihai Lake was stable, ranging from 30 ppm to
41 ppm in the eight sampling sites (Fig. 3). Moreover, the highest
relative abundance of ARGs was observed in S1 and the lowest in S8.
3.3. Subtype abundance of ARGs in the sediment of Daihai Lake
The ARG types were further classified into 111 ARG subtypes across
all samples, ranging from 51 to 67 ARG subtypes per sample. Among all
ARG subtypes, the top 30 of each sampling site were extracted, and a
total of 51 ARG subtypes are presented in SI Fig. 1. The top 30 ARG
subtypes in each sampling site contributed 97.42–99.48% to the total
ARG subtypes abundance. A total of 33 ARG subtypes were shared across
eight sites. Moreover, these ARG subtypes mainly belong to the ami­
noglycoside resistance gene (aac (6′ )-II and aadA), multidrug resistance
(acrA, acrB, acrF, adeB, adeJ, cmeB, emrE, mdtB, mdtC, mdtF, mdtK, mexA,
mexB, mexD, mexE, mexF, mexI, mexT, mexW, mexY, multi­
drug_ABC_transporter, multidrug_transporter, oprM, smeD, smeE, and
TolC), beta-lactam resistance gene (blaOXA-2, blaOXA-226, blaOXA-3, blaOXA-
34, and blaPBP-1A), bacitracin resistance (bacA), MLS (lsa, macA, and
macB), vancomycin resistance gene (vanR and vanS), polymyxin resis­
tance gene (arnA), rifamycin resistance gene (ADP-ribosylating trans­
ferase), sulfonamide resistance gene (sul4), tetracycline resistance gene
(tet35 and tetP), fosmidomycin (rosA and rosB), and kasugamycin
resistance (kasugamycin resistance protein ksgA). At the subtype level,
C. Du et al.
Fig. 3. Percentage abundances of different ARGs in the sediments of Daihai Lake. (Blank represents the ARGs was not detected in sampling site).
bacA, multidrug transporter, macB, and acrB were the dominant ARGs
subtypes, accounting for approximately 64.25–77.63% in each sample.
The bacA gene had the highest abundance, ranging from 6.40 ppm to
17.50 ppm in all samples, constituting approximately 21.65–32.97% of
the total.
3.4. Abundance of MRGs in the sediment of Daihai Lake
The metagenomic analysis demonstrated that 15 MRG types
comprising 146 MRG subtypes were found in the sediment samples.
Resistance genes for Pb, Ni, Hg, W, Zn, Ag, Cr, Fe, As, Cu and multi­
metals shared MRG types in all samples (Fig. 4). The main MRG types
were multimetal resistance genes which accounted for 39.44–43.92% of
the total relative abundance of MRGs. Cu resistance genes with a high
relative abundance of MRGs were discovered, ranging 39.45–43.92%,
followed by MRGs with As resistance (15.54–18.28%), Fe resistance
(5.22–7.29%), and Cr resistance (2.68–4.35%) genes. Only Au resistance
gene was found in S6, with a low value of 0.02%. Furthermore, the
relative abundance of MRGs from the selected sediment samples ranged
from 225 ppm to 459 ppm, with a mean relative abundance of 337 ppm
(Fig. 4). Among all samples, S7 had the highest relative abundance of
MRGs, while S5 had the lowest abundance.
Among all MRG subtypes, the top 30 of each sampling site were
extracted, and a total of 36 MRG subtypes are illustrated in SI Fig.2. The
top 30 MRG subtypes in each sampling site accounted for 94.18–95.70%
of the total MRG subtype abundances. Cu resistance genes (copB, cop-
unnamed, actP, copF, copR, corR, copS, ctpV, copP, and cutO), multi­
metal resistance genes (arsB, arsA, czcC, ruvB, recG, dmeF, golT, copA,
4
Environmental Pollution 302 (2022) 119065
cueA, nczA, and zipB), the Fe resistance gene (acn), As resistance genes
(acr3, arrA, pstB, arsM, arsT, pstC, arsC, and arrB), Cr resistance genes
(chrA and chrF), Ag resistance genes (silA and silP), and Zn resistance
genes (ziaA and zraR/hydH) were the main MRG subtypes. Moreover,
these MRG subtypes belong to resistance genes for Cu, As, and multi­
metals. All the detected MRG subtypes (36 MRG subtypes) were shared
by all samples. CopB, arsB, copA, ruvB, acn, cop-unnamed, actP, acr3,
copF, arrA, pstB, chrA, and copR genes were the most prevalent subtypes,
accounting for 82.81–84.95% in each sample (SI Fig. 2). The copB gene
(Cu resistance gene) had the highest relative abundance in the sample
sites, except for S4 and S5.
3.5. Abundance of MGEs and correlation between resistance genes in the
sediment of Daihai Lake
To investigate the distribution characteristics of MGEs in the sedi­
ment of Daihai Lake, the sequencing data were compared with the
database of mobile genetic elements. Plasmids (91.03–91.82%) were the
most dominant MGEs in all samples, followed by IS (4.14–4.88%) and
ICEs (3.15–3.51%). The relative percentage of other MGEs was
extremely low, with a total percentage of 0.76% (Fig. 5).
Furthermore, network analysis was used to investigate the co-
occurrence patterns of resistance genes subtypes (ARGs and MRGs)
and MGEs (relative abundance > 5e− 5) in the sediment of Daihai Lake.
The results revealed a significant correlation between MGEs and resis­
tance genes (Fig. 6). Furthermore, the entire network consisted of 40
nodes (12 MGEs, 5 MRG subtypes, and 23 ARG subtypes) and 57 edges.
Plasmids were the main MGEs, including unnamed (NZ_CP019912.1,
C. Du et al. Environmental Pollution 302 (2022) 119065

Fig. 4. Percentage abundances of different MRGs in the sediments of Daihai Lake.

Fig. 5. Relative abundance of MGEs in the sediments of Daihai Lake.

NZ_CP020399.1), unnamed1 (NZ_CP022308.1), unnamed2

(NZ_CP024037.1, NZ_CP029357.1), unnamed4 (CP046144.1), p1
(NZ_CP032331.1, NZ_CP032346.1), pG20AB007-2, pAZKH, TSH58_p02,
and pSBC2-1. Among these, unnamed4 (CP046144.1) was significantly
positively associated with the gene of cAMP regulatory protein. The
plasmid of unnamed1 (NZ_CP022308.1) and unnamed2
(NZ_CP029357.1) were positively associated with multiple MRGs. Other
plasmids were significantly positively associated with both ARGs and
MRGs. Fig. 6. Network analysis of the correlation between MGEs, ARGs, and MRGs.

5
C. Du et al.
3.6. Co-occurrence between bacteria, ARGs, and MRGs
Network analysis can reveal the co-occurrence patterns between the
resistome (ARGs and MRGs) and microorganisms. The findings revealed
that there are different co-occurrence relationships between microor­
ganisms and resistance genes for antibiotics and metals in the complex
network structure. The entire network consisted of 127 nodes (75 mi­
croorganisms, 30 MRG subtypes, and 22 ARG subtypes), and 182 edges
(Fig. 7). There are 6 ARG types: multidrug resistance genes (16 ARG
subtypes), Bacitracin (1 ARG subtype), Fosmidomycin (1 ARG subtype),
MLS (2 ARG subtypes), Polymyxin (1 ARG subtype), and beta-lactam (1
ARG subtype). Furthermore, the selected MRG types include multimetal
resistance genes (11 MRG subtypes), Cu (7 MRG subtypes), As (7 MRG
subtypes), Cr (2 ARG subtypes), Fe (1 MRG subtype), Zn (1 MRG sub­
type), and Ag (1 MRG subtype). In total, the target ARGs and MRGs were
significantly positively correlated with the six phyla: Proteobacteria (67
genera), Actinobacteria (4 genus), Bacteroidetes (1 genus), Chloroflexi
(1 genus), Euryarchaeota (1 genus), and Deinococcus-Thermus (1
genus). Proteobacteria was the most common phylum, accounting for
89% of the selected phyla. It was positively correlated with various ARG
and MRG subtypes, particularly MLS resistance gene (macB), multidrug
resistance genes (mdtF, and adeB), multimetal resistance gene (chrC),
and Cu resistance gene (cutO), and it established strong relationships
with various Proteobacteria genera. In addition, the Actinobacteria
phylum (Methanothrix, Mycobacterium, Rhodococcus, and Streptomyces)
was found to be positively correlated with multiple resistance genes. The
genera Methanothrix and Mycobacterium were positively correlated with
ARGs, while Rhodococcus and Streptomyces were positively correlated
with MRGs. Bacteroidetes (Aquiflexum) were positively correlated with
unclassified type (cAMP regulatory protein). From Chloroflexi, Dehalo­
genimonas spp were positively correlated with beta-lactam (blaOXA-34),
polymyxin resistance gene (arnA), multimetal resistance genes (copA),
and As resistance genes (arsM). The genus Deinococcus from the
Deinococcus-Thermus phylum and Micromonospora (phylum Actino­
bacteria) were positively correlated with ARGs linked to multidrug
resistance genes, adeJ, and mexF, respectively. Notably, the selected
Fig. 7. Network analysis of the correlation between bacteria, ARGs, and MRGs.
6
Environmental Pollution 302 (2022) 119065
bacteria may carry different resistance genes, some of which simulta­
neously carry ARGs and MRGs, such as Actinobacteria and Proteobac­
teria. This indicates that these bacterial families may be potential hosts
for resistance genes, and different bacteria play different roles in the co-
occurrence relationship.
4. Discussion
4.1. Distribution of bacterial community composition
In this study, the dispersion, composition, and relationships of
resistomes (ARGs and MRGs) and microbial community structure were
investigated based on a metagenomic analysis of the sediment of Daihai
Lake. The results showed that Proteobacteria, Actinobacteria, Firmi­
cutes, and Bacteroidetes were the main phyla of the bacterial commu­
nity composition in the selected sampling site. A similar phenomenon
was observed in the other lakes (Chakraborty et al., 2020; Pan et al.,
2020). For example, there were >50 bacterial phyla from the given
sediment samples, where the phyla Proteobacteria, Firmicutes, Actino­
bacteria, and Bacteroidetes were the dominant species in Taihu Lake
(Chen et al., 2019b). In addition, these phyla also predominate in other
habitats, such as landfill leachates, wastewater treatment plant, animal
wastes (manure or wastewater), and soil (Gupta et al., 2018; He et al.,
2019; Wang et al., 2021; Zhang et al., 2021). Among the phyla of Pro­
teobacteria, the most dominant phylum in all the samples, has extensive
morphological, physiological, and metabolic diversity and comprises
several known human pathogens (Rizzatti et al., 2017). According to
some studies, Proteobacteria are the most abundant in the composition
of marine microbial communities, including halophilic bacteria. Daihai
Lake is an inland closed water lake with brackish water conducive for
bacterial growth. Following Proteobacteria, Actinobacteria, a ubiqui­
tous and primarily gram-positive bacterial phylum commonly found in
lakes, dominated all sampling sites. The bacteria of this phylum utilize
various resources, including sugars, amino acids, organic acids, and al­
cohols, for development (Sorokin, 2011; Yannarell and Kent, 2009).
Bacteroidetes are the predominant microbial phyla that maintain a
complex and generally beneficial relationship with the host in gut eco­
systems. However, when transferred to different environments, they can
cause severe pathology, such as bacteremia and abscess formation in
various body parts (Wexler, 2007). This group is described as the main
multidrug-resistant bacterial group associated with infections (Gilmore
and Ferretti, 2003). These copiotrophic bacteria (Actinobacteria, Bac­
teroidetes, and Proteobacteria) thrive in nutrient-rich environments and
can rapidly utilize resources. Therefore, these bacteria are widespread
and dominant in nutrient-rich conditions, such as wastewater treatment
plants, soil, landfill leachate, and aquaculture environments (polluted
water bodies) (Fang et al., 2019; Zhang et al., 2021; Zhao et al., 2018).
4.2. Distribution of ARGs and MRGs
A total of 16 types (ranging 30–43 ppm, average relative abundance
of 36.75 ppm) and 111 subtypes of ARGs were discovered in the given
sediment samples. The number and relative abundance of ARGs in
Daihai Lake were less than other lakes (21 ARG types and 321 subtypes
in Lake Tai, 19 ARG types belonging to 133 subtypes with relative
abundances ranging 596–620 ppm in Erhai Lake) (Chen et al., 2019b;
Pan et al., 2020), which indicated that the Daihai basin was moderately
polluted with ARGs. A possible explanation was that Daihai is a conti­
nental closed lake with less exogenous pollution. In addition, the main
ARG types, including resistance genes for MLS, tetracycline,
beta-lactam, and sulfonamide, were detected in the sediment samples,
which was in accordance with the observations of a previous study (Pan
et al., 2020). Among the ARG types, resistance genes for bacitracin were
the most dominant ARG types in all samples. Bacitracin is an extensively
used antibiotic produced by certain strains of Bacillus subtilis and Bacillus
licheniformis. This antibiotic requires some divalent metal ions, such as
C. Du et al.
Zn2+ Mn2+, Co2+, Ni2+, and Cu2+, for its biological activity and is
clinically used as a broad-spectrum antibiotic against gram-positive
bacteria, which cause skin infections (Ming and Epperson, 2002). It is
commonly detected in the hospital environment. Numerous studies have
also reported that bacitracin-resistant genes are found in various natural
habitats (Chen et al., 2016; Chen et al., 2019a; Xi et al., 2018). Among all
ARG subtypes, bacA, which belongs to the resistance gene for bacitracin,
was the dominant ARG subtype at all sampling sites, accounting for
39.44–43.92% of the total relative abundance of ARG subtypes. It was
also found in high relative abundances in soils, deep-sea sediments, and
human feces (Hu et al., 2013). In addition, a total of 15 MRG types and
146 MRG subtypes were discovered in the sediment of Daihai Lake.
Resistance genes for multimetals and Cu were the main types of MRG in
all samples. Heavy metals such as Cu, Pb, Zn, Cd, Al, Fe, and Mn were
detected in the surface sediment samples of Daihai Lake (Jiang et al.,
2014; Sun et al., 2021). The results indicated that atmospheric deposi­
tion, agricultural pollution, and coal ash could be significant sources of
heavy metal pollution (Cu, Pb, Zn, and Cd) in the lake (Jiang et al.,
2014). This may also result in the high relative abundance of MRGs in
the given sediment samples of Daihai Lake. Moreover, it has been
revealed that co-transfer of ARGs and MRGs occurs among microor­
ganisms through MGEs (Li et al., 2017), which has been demonstrated in
various studies (Gupta et al., 2018; Rosewarne et al., 2010; Salam,
2020).
4.3. Co-occurrence between resistance genes and MGEs in the microbial
community
MGEs include IS, ICEs, plasmids, integrators, and transposons, that
can capture and express diverse resistance genes (Gillings et al., 2008).
MGEs have been shown to enhance the transmission and distribution of
ARGs via HGT (Chen et al., 2013). Our results showed a significant
positive correlation between resistance genes and MGEs, particularly
plasmids. Other studies have reported significant correlations between
the abundance of ARGs and the diversity of MGEs (Chen et al., 2019c;
Jiang et al., 2018), suggesting that MGEs play an essential role in the
dissemination and proliferation of ARGs in the environment (Chen et al.,
2013). Furthermore, plasmids were significantly positively associated
with both ARGs and MRGs, which may indicate the co-existence of ARGs
and MRGs in the same plasmid. This could be due to the co-selection of
ARGs caused by heavy metal contamination (Di Cesare et al., 2015;
Imran et al., 2019), which is supported by the fact that there is a certain
degree of heavy metal pollution in the Daihai Lake (Jiang et al., 2014).
In general, microorganisms positively correlated with resistance
genes implying that these phyla may be potential hosts of these resis­
tance genes. Our study found that a total of 52 ARGs and MRGs were
positively correlated with microorganisms such as Proteobacteria and
Actinobacteria. Other studies have found similar results. For example,
ARGs tended to exist in the phylum Proteobacteria (Hu et al., 2013). The
phyla (Proteobacteria and Actinobacteria) have a significantly positive
relationship with various antibiotics and metals (Wang et al., 2019a).
Therefore, as the abundance of these potential host bacteria increases in
the environment, it may promote the spread and transfer of resistance
genes. These results strongly suggested that the composition of micro­
bial communities has a significant impact on the abundance, evolution,
and dissemination of resistomes (ARGs and MRGs) (Che et al., 2019; He
et al., 2019; Song et al., 2019; Wang et al., 2021). Furthermore, several
abiotic factors may also influence resistance genes. TN correlated
negatively with resistance genes for multidrug and rifamycin but posi­
tively with fosfomycin, MLS, and trimethoprim resistance genes (Pan
et al., 2020). The temperature was found to affect the diversity and
abundance of ARGs in the environment (Dunivin and Shade, 2018).
High temperatures favor the mutation of the rpoB gene and can increase
rifampicin resistance in Escherichia coli (Rodríguez-Verdugo et al.,
2013). Daihai Lake is a typical inland lake with a high nutrient con­
centration and considerable temperature variability. These abiotic
7
Environmental Pollution 302 (2022) 119065
factors could also have a significant impact on the resistome. Conse­
quently, in the future, we must delve deeper into the changes and dis­
tribution of resistance genes across seasons, as well as their relationship
with abiotic factors. Furthermore, due to agricultural development,
Daihai Lake has experienced serious environmental problems (water
scarcity and water pollution) (Chun et al., 2020). Agricultural activities
may also influence the distribution and abundance of resistance genes
(Shi et al., 2019). This study demonstrates the relationships between
resistance genes (ARGs and MRGs) and microorganisms in sediment.
However, the potential host, identified by network analysis, is only for
informational purposes. In the future, more in-depth methods will be
required to further investigate hosts that carry resistance genes to
determine the co-occurrence patterns of ARGs, MRGs, and bacteria.
5. Conclusion
Based on the metagenome analysis results obtained in this work, a
wide variety of ARGs and MRGs are present in Daihai Lake. It was also
demonstrated that the lake was an important sink of resistance genes.
Proteobacteria was the main bacterial community component. In total,
16 ARG types and 15 MRG types were identified in the sample, with no
significant changes in the relative abundances of ARGs and MRGs in all
samples. The resistance genes for bacitracin and multidrug remained
abundant in the selected sample. A network analysis of bacteria and ARG
and MRG subtypes revealed that each microorganism may significantly
co-occur with multiple antibiotic and metal resistant genes. These
findings are important for understanding the correlation between
resistance genes and bacteria in the surface sediment samples of Daihai
Lake. Therefore, the seasonal variation of microorganisms and resis­
tance genes should be further explored in future studies to understand
resistance gene behavior in lake habitats.
Author statement
Caili Du: Investigation, Visualization, writing. Fang Yang: Funding
acquisition, Conceptualization, Writing - Review & Editing. Xiaoguang
Li: Funding acquisition, Methodology. Zhonghong Li: Software, Formal
analysis. Jiayue Gao: Software, Formal analysis. Lieyu Zhang: Writing -
Review & Editing, Project administration, Supervision.
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.
Acknowledgments
This research was jointly supported by the National Key Research
and Development Program of China (2019YFC0409205,
2019YFC0409202), National Natural Science Foundation of China
(41907338) and Postdoctoral Science Foundation of China
(2019M660753).
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.envpol.2022.119065.
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