重金属污染土壤中细菌抗生素抗性基因分布特征

doi:10.17520/biods.2024101

生物多样性 ›› 2025, Vol. 33 ›› Issue (3): 24101. DOI: 10.17520/biods.2024101 cstr: 32101.14.biods.2024101

• 研究报告:微生物多样性 • 下一篇

重金属污染土壤中细菌抗生素抗性基因分布特征

仝淼(), 王欢(), 张文双, 王超(), 宋建潇^*()()

西北工业大学生态环境学院, 西安 710129

收稿日期:2024-03-17 接受日期:2024-09-17 出版日期:2025-03-20 发布日期:2024-11-29
通讯作者: *E-mail: sjx@nwpu.edu.cn
基金资助:
国家自然科学基金(41907358);西北工业大学硕士研究生实践创新能力培育基金(PF2024028);中国安能集团第三工程局有限公司横向课题(合同编号2023610109001470)

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

Tong Miao(), Wang Huan(), Zhang Wenshuang, Wang Chao(), Song Jianxiao^*()()

School of Ecology and Environment, Northwestern Polytechnical University, Xi’an 710129, China

Received:2024-03-17 Accepted:2024-09-17 Online:2025-03-20 Published:2024-11-29
Contact: *E-mail: sjx@nwpu.edu.cn
Supported by:
National Natural Science Foundation of China(41907358);Practice and Innovation Funds for Graduate Students of Northwestern Polytechnical University(PF2024028);Open Foundation of China Anneng Group Third Engineering Bureau Co., Ltd(合同编号2023610109001470)

1. 附录.pdf(375KB)

摘要/Abstract

摘要：

重金属作为常见的土壤污染物, 可通过共选择作用诱导土壤微生物群落的抗生素抗性基因(antibiotic resistance genes, ARGs)的产生和扩散, 并促进ARGs在环境中持久存在。本研究建立了不同铜(Cu)浓度和镉(Cd)浓度的土壤微宇宙实验, 基于高通量测序和实时荧光定量PCR技术分析不同重金属浓度和复合污染条件下ARGs的分布特征以及土壤细菌群落对不同浓度Cu、Cd污染的响应, 旨在解析ARGs与土壤细菌群落在重金属污染土壤中的分布特征。此外, 通过探究影响ARGs变化的关键环境因子, 以期找出有效减缓环境中抗生素抗性的传播和扩散现状的途径。研究表明: 高浓度Cu (400 mg/kg)与Cd (1 mg/kg和5 mg/kg)的复合污染显著(P < 0.05)提高了土壤中抗生素抗性基因sul1、intl1、blaVIM的相对丰度, 抗生素抗性基因tetX、tetG对Cd响应最敏感, 低浓度Cd (1 mg/kg)可明显提高tetX、tetG的丰度。此外, Cu、Cd显著(P < 0.05)改变了土壤细菌群落结构, 并且使细菌群落对Cu的响应更加明显。相关性分析和网络分析表明, sul1、tetX、tetM02、blaVIM广泛分布在多个细菌门, 而ARGs的变化与细菌群落(如链霉菌属Streptomyces、慢生根瘤菌属Bradyrhizobium、BIrii41属(Polyangiales)、海无柄孢囊黏细菌属Haliangium等)的变化密切相关, 推测这些细菌可能是携带ARGs的主要宿主。

关键词: 重金属污染, 细菌群落结构, 抗生素抗性基因, 微宇宙, qPCR

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

仝淼, 王欢, 张文双, 王超, 宋建潇 (2025) 重金属污染土壤中细菌抗生素抗性基因分布特征. 生物多样性, 33, 24101. DOI: 10.17520/biods.2024101.

Tong Miao, Wang Huan, Zhang Wenshuang, Wang Chao, Song Jianxiao (2025) Distribution characteristics of antibiotic resistance genes in soil bacterial communities exposed to heavy metal pollution. Biodiversity Science, 33, 24101. DOI: 10.17520/biods.2024101.

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链接本文: https://www.biodiversity-science.net/CN/10.17520/biods.2024101

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

图/表 10

表1 本研究中重金属浓度设置

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

表2 引物序列及扩增条件信息

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

图1 不同浓度Cu和Cd污染土壤抗生素抗性基因(ARGs)和移动基因元件(MGE)绝对丰度(a)与相对丰度(b)。各处理全称见表1。Sul1、tetG、tetM02、blaVIM、tetX和intl1为ARGs和MGE。

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.

图2 不同浓度Cu和Cd污染的土壤细菌16S rRNA基因绝对丰度。各处理全称见表1。不同小写字母表示不同处理间差异显著(P < 0.05)。

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).

图3 不同浓度Cu和Cd重金属污染土壤细菌群落α多样性。各处理全称见表1。

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.

图4 不同浓度Cu和Cd重金属污染土壤细菌前10门水平(a)及属水平(b)群落结构。各处理全称见表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.

图5 土壤细菌群落结构的PCoA分析(a)与土壤抗生素抗性基因(ARGs)、移动基因元件(MGE)和细菌群落(ASVs)网络分析(b)。各处理全称见表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.

图6 属水平前30细菌与ARGs相关性分析(R2表示相关系数, *P < 0.05, **P < 0.01)。Sul1、tetX、blaVIM和intl1为ARGs和MGE。

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.

图7 不同浓度Cu和Cd重金属污染土壤中显著富集的细菌LEfSe分析(a: 进化分支图; b: 线性判别分析(LDA)柱状图)。CK代表对照组, Cu代表单独施加Cu的处理(Cu200、Cu400), Cd代表单独施加Cd的处理(Cd1、Cd5), CuCd代表二者复合污染(Cu200 + Cd1、Cu200 + Cd5、Cu400 + Cd1、Cu400 + Cd5)。当前LDA临界值为3。

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.

表3 重金属Cu、Cd、抗性基因相对丰度相关性分析

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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重金属污染土壤中细菌抗生素抗性基因分布特征

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

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