强氯精对对虾养殖用水中抗生素抗性基因的影响

夏涛涛; 苏浩昌; 胡晓娟; 徐煜; 文国樑; 曹煜成; 余招龙

doi:10.12131/20210361

强氯精对对虾养殖用水中抗生素抗性基因的影响

doi: 10.12131/20210361

夏涛涛^{1, 2,},
苏浩昌²,
胡晓娟²,
徐煜²,
文国樑²,
曹煜成^{1, 2, 3, ,},
余招龙³

1.
上海海洋大学水产与生命学院，上海 201306
2.
中国水产科学研究院南海水产研究所/广东省渔业生态环境重点实验室/农业农村部南海渔业资源开发利用重点实验室，广东广州 510300
3.
岭南现代农业科学与技术广东省实验室茂名分中心，广东茂名 525400

基金项目: 广东省重点领域研发计划 (2021B0202040001)；国家现代农业产业技术体系 (CARS-48)；广东省自然科学基金项目 (2019A1515011618)；广东省现代农业产业技术体系创新团队 (2019KJ149)；中国水产科学研究院基本科研业务费专项资金 (2020TD54, 2020XK02)；中国水产科学研究院南海水产研究所中央级公益性科研院所基本科研业务费专项资金资助 (2021SD08)

详细信息

作者简介:
夏涛涛 (1995—)，男，硕士研究生，研究方向为养殖环境安全。E-mail: 1903096289@qq.com

通讯作者:
曹煜成 (1979—)，男，研究员，博士，研究方向为水产健康养殖和养殖生态环境调控。E-mail: cyc_715@163.com

中图分类号: S 949
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出版历程
- 收稿日期: 2021-12-01
- 修回日期: 2022-03-23
- 录用日期: 2022-03-31
- 网络出版日期: 2022-04-12
- 刊出日期: 2022-12-05

Effect of trichloroisocyanouracic acid on antibiotic resistance genes in aquaculture water of shrimp

XIA Taotao^{1, 2
,},
SU Haochang²,
HU Xiaojuan²,
XU Yu²,
WEN Guoliang²,
CAO Yucheng^{1, 2, 3
, ,},
YU Zhaolong³

1.
College of Fisheries and Life Science, Shanghai Ocean University, Shanghai 201306, China
2.
South China Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences/Key Laboratory of South China Sea Fishery Resources Exploitation & Utilization, Ministry of Agriculture and Rural Affairs/Guangdong Provincial Key Laboratory of Fishery Ecology and Environment, Guangzhou 510300, China
3.
Maoming Branch of Guangdong Laboratory of Lingnan Modern Agricultural Science and Technology, Maoming 525000, China

摘要

摘要: 为探讨渔用氧化剂——强氯精 (三氯异氰脲酸，C₃Cl₃N₃O₃) 对养殖水体环境中抗生素抗性基因 (Antibiotic resistant genes, ARGs) 的去除并控制其传播的可行性，采用实时荧光定量PCR技术，分别对近海水源水、蓄水沉淀池水体、氧化消毒后养殖备用水中的ARGs浓度进行为期29 d的监测分析，其中氧化消毒时的强氯精使用质量浓度为40 mg·L⁻¹。测定的ARGs包括sul1、sul2、tetX、tetM、floR、cmlA和qnrA 7种养殖环境中常见的ARGs。结果显示，sul1、sul2、floR及tetX为上述3种水样中的优势ARGs。近海水源水中ARGs的种类数量及总浓度均最高；蓄水沉淀池水体中ARGs的总浓度低于水源水，sul2和floR的浓度分别较水源水降低了0.86和0.34 lg；经氧化消毒后水体中ARGs的总浓度也有所下降，sul2和floR的浓度与水源水相比分别降低了1.58和1.30 lg。可见，近海水源水是池塘环境中ARGs的主要来源，使用强氯精对其进行氧化消毒，可明显降低水体中常见ARGs水平，有助于防控ARGs在养殖环境中的传播。
- 抗生素抗性基因 /
- 氧化 /
- 养殖水体 /
- 强氯精
Abstract: In order to explore the feasibility of removing and controlling the spread of antibiotic resistant genes (ARGs) by the fishing oxidant trichloroisocyanuric acid (C₃Cl₃N₃O₃) in the aquaculture water environment, we applied the real-time quantitative PCR to monitor and determine the ARGs concentrations in the offshore source water, storage water of sedimentation tank and reserved water after oxidation and disinfection for 29 d. The final concentration of trichloroisocyanuric acid used for oxidation and disinfection was 40 mg·L⁻¹. The target ARGs were commonly found in the aquaculture environments, including sul1, sul2, tetX, tetM, floR, cmlA and qnrA. The results show thatsul1, sul2, floR and tetX were the dominant ARGs among the above mentioned water samples. The number of types and total concentration of ARGs were the highest in offshore source water. The total concentrations of ARGs in the storage water of sedimentation tank were lower than those in the offshore source water, especially that the concentrations of sul2 andfloR were 0.86 and 0.34 lg lower than those in the offshore source water, respectively. After the oxidation and disinfection by trichloroisocyanuric acid, the total concentrations of ARGs in the reserved water decreased, and compared with the offshore source water, the concentrations of sul2 and floR decreased by 1.58 and 1.30 lg, respectively. The results indicate that offshore source water is the main source of ARGs in the aquaculture environment. Oxidation and disinfection treatment on offshore source water with trichloroisocyanuric acid can reduce the concentrations of common ARGs in the aquaculture water significantly, which is helpful to prevent and control the spread of ARGs in aquaculture environments.
- Antibiotic resistance genes /
- Oxidantion /
- Aquaculture water /
- Trichloroisocyanouracic acid

HTML全文

图 1 各样品中ARGs总浓度变化

注：不同字母代表显著差异 (P<0.05)。

Figure 1. Total ARGs concentrations in samples

Note: Different letters indicate significant difference (P<0.05).

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图 2 不同时刻相同采样点各种优势ARGs的浓度

Figure 2. Concentrations of various dominant ARGs at same sampling point at different time

下载: 全尺寸图片幻灯片

图 3 3次取样时ARGs总浓度对比

Figure 3. Comparison of total ARGs concentrations for three samplings

下载: 全尺寸图片幻灯片

表 1 qPCR 反应体系

Table 1. qPCR reaction solution

qPCR反应试剂 qPCR reaction solution	各组分加样量 Amount of usage/μL
荧光定量染料 TB^® Premix Ex Taq™ (Tli RNaseH Plus) (2×)	5
前置引物 (50 μmol·L^‒1) Forward primer (50 μmol·L^‒1)	0.04
后置引物 (50 μmol·L^‒1) Reverse primer (50 μmol·L^‒1)	0.04
样品DNA Template^a	2
超纯水 dd H₂O	2.92
体系总量 Total	10
注：a. 每次定量均设置阳性对照和阴性对照，阳性对照为建立标曲所用标准质粒，阴性对照为dd H₂O。 Note: a. Positive control and negative control were set for each run. The positive control was the standard plasmid used to establish the standard curve, and the negative control was dd H₂O.

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表 2 本研究中qPCR 所需引物

Table 2. Primers used for quantitative PCR in this study

基因 Gene	引物对 Primer pair	序列 (5'—3') Sequence (5'−3')	退火温度 Annealing temperature/℃	片段大小 Amplicon size/bp	参考文献 Reference
sul1	FW	CGCACCGGAAACATCGCTGCAC	62	163	[20]
sul1	RV	TGAAGTTCCGCCGCAAGGCTCG	62	163	[20]
sul2	FV	TCCGGTGGAGGCCGGTATCTGG	62	191	[20]
sul2	RV	CGGGAATGCCATCTGCCTTGAG	62	191	[20]
tetX	FW	AGCCTTACCAATGGGTGTAAA	55	280	[21]
tetX	RV	TTCTTACCTTGGACATCCCG	55	280	[21]
cmlA	FW	GCCAGCAGTGCCGTTTAT	55	158	[22]
cmlA	RV	GGCCACCTCCCAGTAGAA	55	158	[22]
floR	FW	CGGTCGGTATTGTCTTCACG	56	171	[22]
floR	RV	TCACGGGCCACGCTGTAT	56	171	[22]
qnrA	FW	AGGATTTCTCACGCCAGGATT	57	124	[22]
qnrA	RV	CCGCTTTCAATGAAACTGCA	57	124	[22]
tetW	FW	GAGAGCCTGCTATATGCCAGC	55	168	[23]
tetW	RV	GGGCGTATCCACAATGTTAAC	55	168	[23]
注：FW. 上游引物；RV. 下游引物。 Note: FW. Forward primer; RV. Reverse primer.

下载: 导出CSV

表 3 样品中环境因子检测结果

Table 3. Monitoring results of environmental factors in samples mg·L⁻¹

检测样品 Sample	固体悬浮物 SS	氨氮 NH₃-N	硝酸盐氮 NO₃ ⁻-N	亚硝酸盐氮 NO₂ ⁻-N	无机氮 IN	总氮 TN	磷酸盐 PO₄ ³⁻	化学需氧量 COD
SY1	36	0.019	0.012	<0.003	0.034	1.18	0.15	22.2
SY2	9	0.014	<0.006	<0.003	0.016	1.15	0.023	11.2
SY3	10	0.025	<0.006	<0.003	0.025	0.67	0.017	9.2
XSC1	2	0.037	<0.006	0.003	0.043	0.39	0.069	2.9
XSC2	12	0.009	0.009	<0.003	0.019	0.43	0.063	3.2
XSC3	9	<0.006	0.011	<0.003	0.015	0.75	0.074	4.9
XD1	4	<0.006	0.25	<0.003	0.255	0.55	0.047	0.8
XD2	2	0.008	0.311	<0.003	0.322	0.81	0.026	<0.5
XD3	6	<0.006	0.009	<0.003	0.013	0.69	0.017	<0.5
注：<. 检测结果小于检出限以，加粗数值代表检出限。 Note: <. Detection result is less than the detection limit, and value in bold represent the detection limit.

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表 4 样品中ARGs的Ct值检测结果

Table 4. Ct value of ARGs in samples

抗生素抗性基因 ARG	Ct值 Ct value
抗生素抗性基因 ARG	样品 Sample	空白 Blank
sul1	19.24~25.18	29.33~29.85
sul2	12.4~17.5	26.53~27.39
floR	26.99~30.32	33.2~33.93
cmlA	31.21~34.25	33.65~34.79
tetw	29.02~31.8	30.21~30.48
tetX	23.19~30.76	31.7~32.36
qnrA	—	33.61~34.05

下载: 导出CSV

表 5 沉淀及氧化处理对水体中ARGs去除量比较

Table 5. Comparison of removal effects of ARGs in water by　　　　　　　sedimentation and oxidation treatment　　　　lg

处理　 Treatment	总 ARGs Total ARGs	floR	sul1	sul2	tetX
沉淀 Precipitation	0.86^*	0.34	0.17	0.86^*	−0.32
氧化 Oxidation	1.58^*	0.71^*	0.94^*	1.58^*	1.30^*
注：. 表示处理后较水源水中ARGs浓度的差异性显著 (P<0.05)。 Note: . There is a significant difference in ARGs concentrations in the treated water compared with that in the source water (P<0.05).

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