Effect of trichloroisocyanouracic acid on antibiotic resistance genes in aquaculture water of shrimp
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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值均最高；蓄水沉淀池水体中ARGs的总浓度低于水源水中的，sul2和floR的浓度分别较水源水降低了0.86 log和0.34 log；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 log和1.30 log。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。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 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 that sul1, 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 the concentrations of sul2 and floR were 0.86 log and 0.34 log lower than those in the offshore source water, respectively. After 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 log and 1.30 log, 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 the aquaculture environments.
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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
图 3 3次取样时总ARGs总浓度对比
Figure 3. Comparison of total ARGs concentrations for three samplings
表 1 qPCR 反应体系
Table 1. qPCR reaction solution
qPCR反应试剂及器材
qPCR reaction solution各组分加样量
Among of usage实时定量PCR仪 Real-time qPCR meter qTOWER3 荧光定量染料 TB® Premix Ex Taq™ (Tli RNaseH Plus) (2×) 5 μL 前置引物 (50 μmol·L‒1) Forward Primer (50 μmol·L‒1) 0.04 μL 后置引物 (50 μmol·L‒1) Reverse Primer (50 μmol·L‒1) 0.04 μL 样品DNA Template a 2 μL 超纯水dd H2O 2.92 μL 体系总量 Total 10 μL 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
Referencesul1 FW CGCACCGGAAACATCGCTGCAC 62 163 [20] RV TGAAGTTCCGCCGCAAGGCTCG sul2 FV TCCGGTGGAGGCCGGTATCTGG 62 191 [20] RV CGGGAATGCCATCTGCCTTGAG tetX FW AGCCTTACCAATGGGTGTAAA 55 280 [21] RV TTCTTACCTTGGACATCCCG cmlA FW GCCAGCAGTGCCGTTTAT 55 158 [22] RV GGCCACCTCCCAGTAGAA floR FW CGGTCGGTATTGTCTTCACG 56 171 [22] RV TCACGGGCCACGCTGTAT qnrA FW AGGATTTCTCACGCCAGGATT 57 124 [22] RV CCGCTTTCAATGAAACTGCA tetW FW GAGAGCCTGCTATATGCCAGC 55 168 [23] RV GGGCGTATCCACAATGTTAAC 注：FW.上游引物；RV.下游引物。 Note: FW. Forward primer; RV. Reverse primer.
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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 −-NSY1 36 0.019 0.012 <0.003 SY2 9 0.014 <0.006 < SY3 10 0.025 < < XSC1 2 0.037 < 0.003 XSC2 12 0.009 0.009 < XSC3 9 <0.006 0.011 < XD1 4 < 0.25 < XD2 2 0.008 0.311 < XD3 6 < 0.009 < 检测样品
Sample无机氮
IN总氮
TN磷酸盐
PO4 3−化学需氧量
CODSY1 0.034 1.18 0.15 22.2 SY2 0.016 1.15 0.023 11.2 SY3 0.025 0.67 0.017 9.2 XSC1 0.043 0.39 0.069 2.9 XSC2 0.019 0.43 0.063 3.2 XSC3 0.015 0.75 0.074 4.9 XD1 0.255 0.55 0.047 0.8 XD2 0.322 0.81 0.026 <0.5 XD3 0.013 0.69 0.017 < 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, value in bold font represent 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
ARGCt值 Ct value 样品
Sample空白
Blanksul1 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
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表 5 沉淀及氧化处理对水体中ARGs去除量比较
Table 5. Removal effects of ARGs in water by sedimentation and oxidation treatment
处理
Treatment总 ARGs
Total ARGsfloR 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水平的差异性显著 (P<0.05)。 Note: *. Indicates that there is a significant difference in the level of ARGs in the treated water compared with that in the source water (P<0.05).
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