典型场地土壤剖面中抗生素及其抗性基因的分布特征

doi:10.3969/j.issn.1000-5641.2024.06.007

摘要/Abstract

摘要：

场地土壤中存在多种环境污染物, 严重威胁城市土壤和地下水安全. 而深层土壤中的新型污染物研究较少, 例如抗生素和抗生素抗性基因 (ARGs), 需要及时监测和治理. 本研究选取河北邯郸明芳钢铁场地为研究区域, 采集了3根4 m的土壤柱. 采用超高效液相色谱-串联质谱仪检测了其中四大类 (磺胺类、氟喹诺酮类、大环内酯类、四环素类) 共17种抗生素的含量, 运用宏基因组测序技术研究了不同深度土壤中ARGs的种类、相对丰度和贡献物种. 研究结果表明, 总抗生素污染浓度范围在2593.2 ~ 4279.5 ng/g之间, 其中磺胺类抗生素 (SAs) 为166.1 ~ 1103.8 ng/g, 氟喹诺酮类抗生素 (FQs) 为993.3 ~ 1330.4 ng/g, 大环内酯类抗生素 (MLs) 为4.0 ~ 104.8 ng/g, 四环素类抗生素 (TCs) 为1269.5 ~ 2077.6 ng/g, 与其他区域土壤中抗生素浓度相比, 污染处于中度水平. 土壤剖面中, SAs在浅层土壤 (0—150 cm) 中占比高于深层土壤 (150—400 cm). MLs在深层土壤 (350—400 cm) 中有较多的累积. TCs和FQs的占比在各土壤层中均较高, 是检测到的主要抗生素类别. 四大类抗生素对应的ARGs丰度均随土壤深度增加而减少, 其中大环内酯类ARGs相对丰度最大, 检出频率为0.92 × 10⁵ ~ 1.44 × 10⁵. 物种贡献度统计结果表明, 放线菌门 (Actinobacteria) 对四大类抗生素抗性基因的丰度均具有最大的贡献. 综上, 本研究揭示了抗生素在场地土壤中的分布迁移特征, 而ARGs在深层土壤中的污染不容忽视, 可能会污染地下水, 增加抗生素耐药菌的传播.

关键词: 钢铁冶炼厂场地, 土壤柱, 抗生素, 抗生素抗性基因, 宏基因组测序

Abstract:

The presence of a variety of environmental contaminants in the soils of steelworks seriously threatens the safety of urban soil and groundwater. Emerging pollutants in deep soils, such as antibiotics and antibiotic resistance genes (ARGs), have not been studied extensively and require timely monitoring and treatment. In this study, the Mingfang Iron and Steel Plant in Handan, Hebei Province, was selected as the research area, and three 4-meter soil columns were collected. The contents of 17 antibiotics in four categories (sulfonamides, fluoroquinolones, macrolides, and tetracyclines) were detected by ultra-performance liquid chromatography-tandem mass spectrometry. The types and abundances of ARGs in the soil at different depths were investigated by metagenomic sequencing. The results showed that the total antibiotic concentrations ranged from 2593.2 to 4279.5 ng/g, among which sulfonamide antibiotics (SAs) were 166.1 to 1103.8 ng/g, fluoroquinolone antibiotics (FQs) were 993.3 to 1330.4 ng/g, macrolide antibiotics (MLs) ranged from 4.0 to 104.8 ng/g, and tetracycline antibiotics (TCs) ranged from 1269.5 to 2077.6 ng/g, with mild to moderate levels of contamination. Soil profile analysis demonstrated that the percentage of SAs were higher in the topsoils (0—150 cm) than in the deep soils (150—400 cm). Contrastingly, MLs accumulated more in deep soils (350—400 cm). The percentages of TCs and FQs were higher in all soil layers and they were the main antibiotic classes detected. The abundance of ARGs corresponding to the four classes of antibiotics decreased with increasing soil depth. The relative abundance of macrolide ARGs was the highest, with the read number of the detected sequences ranging from 0.92 × 10⁵ to 1.44 × 10⁵. The statistical chart of species contributions indicated that Actinobacteria made the greatest contribution to the abundance of ARGs in all four major categories. In conclusion, this study revealed the distribution and migration characteristics of antibiotics in the site soils. Deep soil pollution by ARGs is a matter of concern as it may lead to groundwater contamination and the increased distribution of antibiotic-resistant bacteria.

Key words: steelworks site, soil column, antibiotics, antibiotic resistance genes, metagenomics sequencing

中图分类号:

关铭茵, 刘欣然, 刘敏, 杨静, 谢泽莹, 张庆玲, 李倩. 典型场地土壤剖面中抗生素及其抗性基因的分布特征[J]. 华东师范大学学报（自然科学版）, 2024, 2024(6): 74-85.

Mingyin GUAN, Xinran LIU, Min LIU, Jing YANG, Zeying XIE, Qingling ZHANG, Qian LI. Characterization of the distribution of antibiotics and their resistance genes in soil profiles of a typical site[J]. Journal of East China Normal University(Natural Science), 2024, 2024(6): 74-85.

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抗生素种类	英文名称	化学式	分子量
磺胺嘧啶 (SD)	sulfadiazine	C₁₀H₁₀N₄O₂S	250.28
磺胺吡啶 (SP)	sulfapyridine	C₁₁H₁₁N₃O₂S	249.29
磺胺甲恶唑 (SMX)	sulfamethoxazole	C₁₀H₁₁N₃O₃S	253.28
磺胺噻唑 (ST)	sulfathiazole	C₉H₉N₃O₂S₂	255.32
磺胺甲基嘧啶 (SM)	sulfamerazine	C₁₁H₁₂N₄O₂S	264.30
磺胺二甲嘧啶 (SMT)	sulfamethazine	C₁₂H₁₄N₄O₂S	278.33
磺胺喹恶啉 (SQ)	sulfaquinoxaline	C₁₄H₁₂N₄O₂S	300.34
诺氟沙星(NFC)	norfloxacin	C₁₆H₁₈FN₃O₃	319.33
环丙沙星 (CFC)	ciprofloxacin	C₁₇H₁₈FN₃O₃	331.34
恩诺沙星 (EFC)	enrofloxacin	C₁₉H₂₂FN₃O₃	359.40
氧氟沙星 (OFC)	ofloxacin	C₁₈H₂₀FN₃O₄	361.37
四环素 (TC)	tetracycline	C₂₂H₂₄N₂O₈	444.45
土霉素 (OTC)	oxytetracycline	C₂₂H₂₄N₂O₉	460.43
强力霉素 (DXC)	doxycyclinehyclate	C₂₂H₂₄N₂O₈	444.44
金霉素 (CTC)	chlorotetracycline	C₂₂H₂₃ClN₂O₈	478.88
红霉素 (ETM)	erythromycin	C₃₇H₆₇NO₁₃	733.94
罗红霉素 (RTM)	roxithromycin	C₄₁H₇₆N₂O₁₅	837.05

样品	pH值	土壤机械组成/%			总氮/%	总碳/%
样品	pH值	黏土	粉砂	砂	总氮/%	总碳/%
A_0—50	11.7	4.3	93.1	2.7	0.02	2.71
A_100—150	8.0	4.4	85.0	10.6	0.02	1.29
A_200—250	8.0	13.0	85.8	1.2	0.01	0.42
A_350—400	7.6	9.4	89.7	1.0	0.02	0.50
B_0—50	8.1	6.9	92.8	0.4	0.03	1.34
B_100—150	10.9	7.1	92.7	0.2	0.03	1.87
B_200—250	7.2	5.9	91.0	3.2	0.02	0.31
B_350—400	7.6	8.9	87.5	3.6	0.03	0.55
C_0—50	11.3	6.8	75.5	17.7	0.03	4.27
C_100—150	8.5	8.5	91.3	0.2	0.03	1.22
C_200—250	8.5	26.0	74.0	0.0	0.03	0.39
C_350—400	8.4	16.6	83.0	0.5	0.03	0.75

编号	SAs/(ng·g^–1)	FQs/(ng·g^–1)	TCs/(ng·g^–1)	MLs/(ng·g^–1)	平均浓度/(ng·g^–1)	总浓度/(ng·g^–1)
1^[35]	0.50 ~ 276.64		0 ~ 46.10	0.06 ~ 1.51	11.93 ~ 32.86
2^[36]	< 10		1335.24			468.79 ~ 2984.64
3^[37]	53.68 ~ 155.24
4^[38]	3.00 ~ 8.54	28.42 ~ 119.57	ND
5^[39]	2.2 ~ 12.2			0.9 ~ 16.7
6^[40]			80 ~ 6006
7^[40]			84 ~ 1421
8	4.94 ~ 89.09	8.49 ~ 318.10	7.24 ~ 458.77	0.20 ~ 0.57	531.95	38.45 ~ 866.37

土壤层	SAs_ARGs	FQs_ARGs	MLs_ARGs	TCs_ARGs
B1	5.23 × 10³	2.40 × 10⁴	1.44 × 10⁵	1.26 × 10⁵
B2	4.08 × 10³	2.11 × 10⁴	1.05 × 10⁵	0.94 × 10⁵
B3	3.68 × 10³	2.03 × 10⁴	0.92 × 10⁵	0.77 × 10⁵

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