冬季天津临港复合型人工湿地对抗生素抗性基因的去除效果

张欣宇; 邢畅; 王丽平; 贾雪红; 张宇轩; 徐秀丽

doi:10.12153/j.issn.1674-991X.20230632

冬季天津临港复合型人工湿地对抗生素抗性基因的去除效果

doi: 10.12153/j.issn.1674-991X.20230632

张欣宇^{1, 2,},
邢畅^{1, 2},
王丽平^1, ,,
贾雪红^{1, 2},
张宇轩¹,
徐秀丽²

1.
中国环境科学研究院
2.
中国地质大学（北京）海洋学院

基金项目: 中央级公益性科研院所基本科研业务费专项（2022YSKY-03）

详细信息

作者简介:
张欣宇（2000—），女，硕士研究生，主要研究方向为资源与环境，z1920831143@163.com

通讯作者:
王丽平（1974—），女，研究员，主要从事河口与近岸海域环境监测和生态风险评价研究，wanglp@craes.org.cn

中图分类号: X714
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出版历程
- 收稿日期: 2023-08-31

Removal of antibiotic resistance genes by composite constructed wetlands of Tianjin Lingang in Winter

ZHANG Xinyu^{1, 2
,},
XING Chang^{1, 2},
WANG Liping^{1
, ,},
JIA Xuehong^{1, 2},
ZHANG Yuxuan¹,
XU Xiuli²

1.
Chinese Research Academy of Environmental Sciences
2.
School of Ocean Sciences, China University of Geosciences (Beijing)

摘要

摘要:
环境中耐药菌和抗生素抗性基因（ARGs）因抗生素的大量应用而广泛存在，影响抗生素对疾病的治疗效果，对人体健康和生态安全构成威胁。研究表明人工湿地（CWs）能够有效去除ARGs，但目前对于北方冬季复合型CWs对ARGs的去除效果尚不明确。以天津临港复合型的CWs（调节塘+水平潜流湿地+表流湿地工艺）为研究对象，开展冬季其对ARGs去除效果的研究；针对不同功能区采集水体样本，采用高通量荧光定量PCR对水体中16S rRNA基因、ARGs、可移动遗传元件（MGEs）及细菌种群组成进行检测，综合分析CWs对ARGs的去除效果，探讨冬季运行期间影响去除效果的关键因素。结果表明：冬季水体中表征细菌数量的16S rRNA基因绝对丰度为2.70×10⁴~1.41×10⁵拷贝/mL；ARGs总检出率为72.5%，其中floR和sul2并非源于进水。各ARGs在不同功能区的丰度差异明显，不同功能区对ARGs的去除效果也存在明显差异。整体来看，CWs对氨基糖苷类抗性基因和多重耐药基因的去除效果最好，总绝对丰度去除率分别为85.59%、47.78%，总相对丰度去除率分别为97.09%、89.44%；对β-内酰胺类抗性基因去除效果最差，总绝对丰度和相对丰度去除率分别为−404.40%、−2.01%。调节塘、水平潜流湿地、表流湿地对ARGs总绝对丰度去除率分别为38.05%、−7.78%和−2.41%；总相对丰度去除率分别为75.02%、−45.60%和−7.75%，不同功能区的去除效果表现为调节塘>表流湿地>水平潜流湿地，其中调节塘对除四环素类抗性基因外的其他ARGs的绝对丰度均有较好的去除效果，水平潜流湿地对磺胺类抗性基因去除效果较好，表流湿地对大环内酯类抗性基因有一定的去除效果。冬季低温、MGEs、不同工艺类型功能区及其运行时间是ARGs去除效果的关键影响因素，ARGs对细菌宿主的非选择性促进了其在天津临港CWs系统中各种细菌类群间的迅速传播。建议今后加强CWs对新污染物ARGs去除效果的优化技术研究。
- 抗生素抗性基因（ARGs） /
- 可移动遗传元件（MGEs） /
- 细菌种群 /
- 人工湿地（CWs） /
- 冬季
Abstract:
Drug-resistant bacteria and antibiotic resistance genes (ARGs) exist widely in the environment due to the extensive application of antibiotics, which affect the therapeutic effect of antibiotics on diseases and pose a great threat to human health and ecological security. Studies have shown that constructed wetlands (CWs) can effectively remove ARGs, but the effect of composite CWs on ARGs removal in northern China in winter is still unclear. Tianjin Lingang CWs, a compound of regulating pond, horizontal subsurface flow wetland and surface flow wetland, was used as the research object to study the removal effect of ARGs in winter. Water samples were collected from different functional zones, and 16S rRNA genes, ARGs, mobile genetic elements (MGEs) and bacterial population composition in water were detected by high-throughput quantitative PCR. The removal effect of ARGs was comprehensively analyzed, and the key factors affecting the removal effect during winter operations were discussed. The results showed that the absolute abundance of 16S rRNA gene was 2.70×10⁴-1.41×10⁵ copies/mL. The total detection rate of ARGs was 72.5%, in which floR and sul2 did not originate from influent water. The abundance of ARGs in different functional zones was significantly different, and the removal effect of ARGs in different functional zones was also significantly different. Overall, CWs in Tianjin Lingang had the best removal effect on aminoglycoside resistance genes and multi-drug resistance genes, with total absolute abundance removal rates of 85.59% and 47.78%, and total relative abundance removal rates of 97.09% and 89.44%, respectively. The removal efficiency of β-lactam resistance genes was the worst, and the total absolute abundance and relative abundance removal rates were −404.40% and −2.01%, respectively. The removal rates of total absolute abundance of ARGs were 38.05%, −7.78% and −2.41% in regulating pond, horizontal subsurface flow wetland and surface flow wetland, and the total relative abundance removal rates were 75.02%, −45.60% and −7.75%, respectively. The removal effects of different functional zones were as follows: the regulating pond > surface flow wetland > horizontal subsurface flow wetland, in which the regulating pond had a better removal effect for the absolute abundance of other ARGs except tetracycline resistance genes, the horizontal subsurface flow wetland had a better removal effect for sulfonamides resistance genes, and the surface flow wetland had a certain removal effect for macrolide resistance genes. Low temperature, MGEs, functional zones of different process types and operation time were the key factors affecting the removal effect of ARGs. The non-selectivity of ARGs to bacterial hosts promoted the rapid spread of ARGS among various bacterial groups in Tianjin Lingang CWs system. It is suggested to strengthen the optimization technology research to improve the removal effect of new pollutant ARGs by CWs.
- antibiotic resistance genes (ARGs) /
- mobile genetic elements(MGEs) /
- bacterial population /
- constructed wetlands(CWs) /
- winter

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图 1 天津临港CWs不同功能区采样点位布设

Figure 1. Layout of sampling stations in different functional zones of Tianjin Lingang CWs

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图 2 天津临港CWs不同功能区采样点16S rRNA基因绝对丰度

Figure 2. Absolute abundances of 16S rRNA genes at the sampling stations in different functional zones of Tianjin Lingang CWs

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图 3 不同功能区采样点ARGs绝对丰度和相对丰度

Figure 3. Absolute abundance and relative abundance of ARGs at each sampling point in different functional zones

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图 4 天津临港CWs不同功能区对不同ARGs的去除率（续）

Figure 4. Removal rate of different ARGs in different functional zones of Tianjin Lingang CWs (continued)

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图 5 不同功能区MGEs绝对丰度分布

Figure 5. MGEs absolute abundance distribution at different functional zones

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图 6 天津临港CWs中主要ARGs绝对丰度与MGEs绝对丰度相关性

注：**表示P<0.01；*表示P<0.05。

Figure 6. Correlation between absolute abundances of main ARGs and MGEs in Tianjin Lingang CWs

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图 7 各采样点属水平上的细菌组成

Figure 7. Bacterial composition at genus level at each sampling site

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图 8 细菌与ARGs的Network分析

注：红色线条代表正相关，绿色线条代表负相关。

Figure 8. Network analysis of bacteria and ARG

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表 1 目标基因种类及名称

Table 1. Target gene species and name

种类	目标基因
β-内酰胺类	ampC、blaCMY、blaOXA10、blaTEM、cphA
磺胺类	sul1、sul2
氯霉素类	cmlA1
多重耐药基因	floR、qacEdelta1、qacH
大环内酯类	ermF、ermT、ereA
四环素类	tetB、tetG、tetX、tetM
氨基糖苷类	aac(6')-I1、aadA、aadA2、strA、strB、aac(6')-Ib
MGEs	intI-1 、cIntI-1、tnpA-01、tnpA-02、tnpA-03、tnpA-04、tnpA-05、tnpA-07、IS613、Tp614

下载: 导出CSV

表 2 各采样点ARGs检出率

Table 2. Detection rate of ARGs of each sampling point 　

ARGs		T1	T2	T3	T4	T5	T6	T7	T8	T9	T10	检出率/%
β-内酰胺类	ampC	+	+	+	+	+	+	+	+	+	+	100
	blaOXA10	+	+	+	+	+	+	−	−	+	−	70
	blaTEM	+	+	+	+	+	+	+	+	+	+	100
	cphA	+	+	+	+	+	+	+	+	+	+	100
磺胺类	sul1	+	+	+	+	+	+	+	+	−	+	90
磺胺类	sul2	−	−	+	+	+	+	−	+	−	−	50
氯霉素类	cmlA1	+	+	+	+	+	+	+	+	+	+	100
多重耐药基因	floR	−	+	−	+	+	+	+	+	+	+	80
	qacEdelta1	+	+	+	+	+	+	+	+	+	+	100
	qacH	+	+	+	+	+	+	+	+	+	+	100
大环内酯类	ereA	+	+	+	+	+	+	+	+	+	+	100
	ermF	+	+	+	+	+	+	−	+	+	+	90
	ermT	+	+	+	+	+	+	+	+	+	+	100
四环素类	tetG	+	+	+	+	+	+	+	−	−	+	80
四环素类	tetX	+	+	+	+	+	+	+	+	+	+	100
氨基糖苷类	aac(6')-Ib	+	+	+	+	+	+	+	+	+	+	100
	aadA	+	+	+	+	+	+	+	−	+	+	90
	aadA2	+	+	+	+	+	+	+	+	+	+	100
	strB	+	+	+	+	+	+	+	+	−	+	90
注：+表示检出；−表示未检出。

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表 3 ARGs绝对丰度与16S rRNA基因绝对丰度的相关系数

Table 3. Correlation coefficient between absolute abundance of ARGs and the absolute abundance of 16S rRNA gene

ARGs		16S rRNA
β-内酰胺类	cphA	0.685*
	blaTEM	0.758*
	blaOXA10	−0.436
	ampC	0.285
磺胺类	sul1	0.367
磺胺类	sul2	0.900
氯霉素类	cmlA1	−0.689*
多重耐药基因	qacH	0.079
	qacEdelta1	−0.139
	floR	0.881**
大环内酯类	ermT	0.176
	ermF	0.533
	ereA	0.624
四环素类	tetX	0.394
四环素类	tetG	0.095
氨基糖苷类	strB	−0.321
	aadA	0.091
	aadA2	−0.018
	aac(6′)-Ib	0.217
总ARGs		0.869**
注：*表示P<0.01；表示P<0.05。

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表 4 不同类型CWs对ARGs去除效果

Table 4. Removal effect of ARGs by different types of constructed wetlands %　

人工湿地类型	16S rRNA	磺胺类抗性基因			四环素类抗性基因				β-内酰胺类抗性基因		大环内酯类抗性基因		氨基糖苷类抗性基因	甲氧苄啶类抗性基因	多重耐药基因
人工湿地类型	16S rRNA	sul1	sul2	sul3	tetO	tetX	tetM	txtB	blaOXA-48	blaTEM	ermB	ermC	aac(6')-Ib	dfrA	qnrA
水平潜流湿地^[66]	32.0	96.9	95.2		−18.1								55.9	−145.6	91.5
表流湿地+潜流湿地^[67]		73.9	75.9		80.2				去除效果差
4个表流湿地+ 1个潜流湿地^[68]		97.2	95.4	83.4	98.9		99.8	99.6			99.1	43.1
表流湿地^[69]		74.0	91.0		90.0	98.0
水平潜流湿地^[70]	16.5~79.3	26.5~79.3			8.4~90.1						20.6~88.0
潮汐-复合流湿地^[71]		82.82~85.66			93.10~94.87

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