Distribution characteristics of antibiotic resistance genes in soil bacterial communities exposed to heavy metal pollution

doi:10.17520/biods.2024101

Abstract

Abstract:

Aims: This work investigates how different soil concentrations of cadmium (Cd) and copper (Cu) affect bacterial community structure and bacterial antibiotic resistance genes (ARGs). Furthermore, we explored key environmental factors that influence ARGs expression, to identify effective approaches to mitigate the spread of antibiotic resistance within environment.
Methods: We established a soil microcosm experiment using soil containing different concentrations of Cu and Cd. The relative expression of ARGs in the soil bacteria was determined by quantitative polymerase chain reaction (qPCR) experiments. We performed genomic sequencing/16S rRNA sequencing using the Illumina MiSeq system to assess the bacterial diversity of soil samples.
Results: High concentrations of Cd and Cu contamination significantly increased the relative abundance of the ARGs, such as sul1, intl1, and blaVIM. The ARGs tetX and tetG were most sensitive to Cd, as their abundance significantly increased even at low concentrations of Cd. In addition, Cu and Cd clearly altered soil bacterial community structure. Overall, the change in bacterial diversity was more pronounced in response to Cu than to Cd. Correlation analysis and network analysis showed that ARGs detected in this study, such as sul1, tetX, tetM02, and blaVIM, were widely distributed across multiple bacterial phyla. Changes in ARG profiles were closely associated with variations in bacterial communities (e.g. Streptomyces, Bradyrhizobium, genus BIrii41 (Polyangiales), and Haliangium), which indicate that these genera are the potential hosts of these resistance genes.
Conclusion: Our work provides direct evidence that heavy metals like Cu and Cd may co-select for ARGs in soil bacterial communities, which poses a potential health risk to humans.

Key words: heavy metal pollution, bacterial community structure, antibiotic resistance genes, microcosm, qPCR

Tong Miao, Wang Huan, Zhang Wenshuang, Wang Chao, Song Jianxiao. Distribution characteristics of antibiotic resistance genes in soil bacterial communities exposed to heavy metal pollution[J]. Biodiv Sci, 2025, 33(3): 24101.

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URL: https://www.biodiversity-science.net/EN/10.17520/biods.2024101

https://www.biodiversity-science.net/EN/Y2025/V33/I3/24101

Figures/Tables 10

Table 1 Concentrations of heavy metals used in this study

处理名称 Treatment	Cu (mg/kg)	Cd (mg/kg)
CK	0	0
Cu200	200	0
Cu400	400	0
Cd1	0	1
Cd5	0	5
Cu200 + Cd1	200	1
Cu200 + Cd5	200	5
Cu400 + Cd1	400	1
Cu400 + Cd5	400	5

Table 2 Primer sequences and amplification conditions

目标基因 Target genes	引物(5°-3°) Primers (5°-3°)	片段长度 Fragment length (bp)	退火温度 Annealing temperature (℃)	参考文献 References
sul1	F: CGGCGTGGGCTACCTGAACG R: GCCGATCGCGTGAAGTTCCG	433	60	Frank et al, 2007
tetG	F: GCAGAGCAGGTCGCTGG R: CCYGCAAGAGAAGCCAGAAG	134	54	Aminov et al, 2001
tetM02	F: ACAGAAAGCTTATTATATAAC R: TGGCGTGTCTATGATGTTCAC	171	52	Aminov et al, 2001
blaVIM	F: CAGATTGCCGATGGTGTTTGG R: AGGTGGGCCATTCAGCCAGA	523	55	Ktari et al, 2006
tetX	F: CAATAATTGGTGGTGGACCC R: TTCTTACCTTGGACATCCCG	468	55	Ng et al, 2001
intl1	F: CTGGATTTCGATCACGGCACG R: ACATGCGTGTAAATCATCGTCG	473	60	Frank et al, 2007
16S V3	F: CCTACGGGAGGCAGCAG R: ATTACCGCGGCTGCTGG	193	55	Aminov et al, 2001
16S V3-V4	F: ACTCCTACGGGAGGCAGCA R: GGACTACHVGGGTWTCTAAT	480	53	Claesson et al, 2009

Fig. 1 The absolute and relative abundance of antibiotic resistance genes (ARGs) and mobile genetic element (MGE) in different concentration of Cu and Cd polluted soils. The full names of all treatments are listed in Table 1. Sul1, tetG, tetM02, blaVIM, tetX, and intl1 are ARGs and MGE.

Fig. 2 Absolute abundance of soil bacteria (16S rRNA gene) in different concentration of Cu and Cd polluted soils. The full names of all treatments are listed in Table 1. Different lowercase letters indicate a significant difference between treatments (P < 0.05).

Fig. 3 Bacterial α diversity in different concentration of Cu and Cd polluted soils. The full names of all treatments are listed in Table 1.

Fig. 4 Community structure of top 10 soil bacteria in different concentration of Cu and Cd polluted soils at the phylum (a) and genus level (b). The full names of all treatments are listed in Table 1.

Fig. 5 PCoA analysis of soil bacterial community structure (a) and network analysis of soil antibiotic resistance genes (ARGs), mobile genetic elements (MGE) and bacterial communities at ASV level (b).The full names of all treatments are listed in Table 1.

Fig. 6 Correlation analysis between the top 30 bacteria genera and ARGs (R2 represents the correlation coefficient. * P < 0.05, ** P < 0.01). Sul1, tetX, blaVIM, and intl1 are ARGs and MGE.

Fig. 7 LEfSe analysis of significantly enriched bacteria in soil under different concentrations of Cu and Cd (a, Cladogram; b, Histogram of linear discriminant analysis (LDA)). CK, Control group; Cu, Treatment with Cu alone (Cu200, Cu400); Cd, Treatment with Cd alone (Cd1, Cd5); CuCd, Combined pollution of Cu and Cd (Cu200 + Cd1, Cu200 + Cd5, Cu400 + Cd1, Cu400 + Cd5). The current LDA threshold is 3.

Table 3 Correlation analysis of relative abundance between Cu, Cd, and ARGs

相对丰度 Relative abundance	Cu	Cd	sul1	tetX	intl1	tetG	tetM02	blaVIM
ARGs	0.265	0.714**	‒0.156	0.497**	‒0.356	‒0.164	0.714**	0.875**
Cu		0.106	‒0.653**	‒0.557**	‒0.621**	0	‒0.228	0.026
Cd			0.182	0.499**	‒0.522**	‒0.358	0.659**	0.77**
sul1				0.42*	0.255	0.004	0.232	‒0.013
tetX					0.337	‒0.179	0.733**	0.632**
intl1						0.185	‒0.086	‒0.213
tetG							‒0.009	‒0.291
tetM02								0.819**

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