Effect of trichloroisocyanouracic acid on antibiotic resistance genes in aquaculture water of shrimp
-
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.
-
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. 
下载: 导出CSV
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.
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 −-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.
下载: 导出CSV
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
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. 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).
下载: 导出CSV
-
[1] 张骞月, 赵婉婉, 吴伟. 水产养殖环境中抗生素抗性基因污染及其研究进展[J]. 中国农业科技导报, 2015, 17(6): 125-134. [2] KYRIAKOU P K, EKBLAD B, KRISTIANSEN P E, et al. Interactions of a class IIb bacteriocin with a model lipid bilayer, investigated through molecular dynamics simulations[J]. BBA-Biomembranes, 2016, 1858(4): 824-835. doi: 10.1016/j.bbamem.2016.01.005 [3] YANG Y, CAO X, LIN H, et al. Antibiotics and antibiotic resistance genes in sediment of Honghu Lake and East Dongting Lake, China[J]. Microb Ecol, 2016, 72(4): 791-801. doi: 10.1007/s00248-016-0814-9 [4] LULIJWA R, RUPIA E J, ALFARO A C. Antibiotic use in aquaculture, policies and regulation, health and environmental risks: a review of the top 15 major producers[J]. Rev Aquacult, 2019, 12(2): 640-663. [5] WANG J, CHU L, WOJNÁROVITS L, et al. Occurrence and fate of antibiotics, antibiotic resistant genes (ARGs) and antibiotic resistant bacteria (ARB) in municipal wastewater treatment plant: an overview[J]. Sci Total Environ, 2020, 744: 140997. doi: 10.1016/j.scitotenv.2020.140997 [6] 叶繁, 冯时欢, 吴佳佳, 等. 养殖虾塘常见耐药菌的分离鉴定与耐药基因检测[J]. 生态环境学报, 2019, 28(9): 1843-1849. [7] 黄志坚, 陈旭凌, 路晓峰, 等. 水产养殖生物和养殖环境细菌鉴定及抗生素抗性基因检测[J]. 中山大学学报 (自然科学版), 2012, 51(6): 92-96. [8] 巴永兵, 娄阳, 赵飞, 等. 水产品批发市场中抗生素抗性基因的污染分析[J]. 食品与生物技术学报 2017, 36(8): 800-806. [9] SU H, LIU S, HU X, et al. Occurrence and temporal variation of antibiotic resistance genes (ARGs) in shrimp aquaculture: ARGs dissemination from farming source to reared organisms[J]. Sci Total Environ, 2017, 607-608: 357-366. doi: 10.1016/j.scitotenv.2017.07.040 [10] SANAWAR H, XIONG Y, ALAM A, et al. Chlorination or monochloramination: Balancing the regulated trihalomethane formation and microbial inactivation in marine aquaculture waters[J]. Aquaculture, 2017, 480: 94-102. doi: 10.1016/j.aquaculture.2017.08.014 [11] 赵晓雨, 苏浩昌, 徐煜, 等. 渔用氧化剂对水源水和池塘水中磺胺类抗性基因sul1的去除作用[J]. 南方水产科学, 2021, 17(03): 46-53. [12] 黎建斌, 陈福艳, 李大列, 等. 三种消毒剂对池塘养殖水中细菌的杀灭效果[J]. 河北渔业, 2018(10): 44-45. doi: 10.3969/j.issn.1004-6755.2018.10.013 [13] 姜叶琴, 姚健萍, 杨万喜. 水产养殖水体处理方法及应用前景综述[J]. 海洋湖沼通报, 2004(3): 99-104. [14] 周孝治, 陈菊芳, 聂湘平, 等. 三氯异氰尿酸在模拟水生态系统中归趋的研究[J]. 生态科学, 2007, 26(4): 361-366. [15] ZHANG T, HU Y, JIANG L, et al. Removal of antibiotic resistance genes and control of horizontal transfer risk by UV, chlorination and UV/chlorination treatments of drinking water[J]. Chem Eng J, 2019, 358: 589-597. doi: 10.1016/j.cej.2018.09.218 [16] 刘雨昊, 郭宁, 刘梦梦, 等. 污水中抗生素抗性基因去除技术研究进展[J]. 环境污染与防治, 2021, 43(6): 764-771. [17] SOUSA J M, MACEDO G, PEDROSA M, et al. Ozonation and UV254 nm radiation for the removal of microorganisms and antibiotic resistance genes from urban wastewater[J]. J Hazard Mater, 2017, 323(Pt A): 434-441. [18] 张治国, 李斌绪, 李娜, 等. 污水深度处理工艺对抗生素抗性菌和抗性基因去除研究进展[J]. 农业环境科学学报, 2018, 37(10): 2091-2100. doi: 10.11654/jaes.2018-0098 [19] YOON Y G, CHUNG H J, DI D Y W, et al. Inactivation efficiency of plasmid-encoded antibiotic resistance genes during water treatment with chlorine, UV, and UV/H2O2[J]. Water Res, 2017, 123: 783-793. doi: 10.1016/j.watres.2017.06.056 [20] PEI R, KIM S C, CARLSON K H, et al. Effect of river landscape on the sediment concentrations of antibiotics and corresponding antibiotic resistance genes (ARG)[J]. Water Res, 2006, 40(12): 2427-2435. doi: 10.1016/j.watres.2006.04.017 [21] DIEHL D L, LAPARA T M. Effect of temperature on the fate of genes encoding tetracycline resistance and the integrase of Class 1 integrons within anaerobic and aerobic digesters treating municipal wastewater solids[J]. Environ Sci Technol, 2010, 44: 9128-9133. doi: 10.1021/es102765a [22] VIEN LE T M, MINH N N, THUONG T C, et al. The co-selection of fluoroquinolone resistance genes in the gut flora of Vietnamese children[J]. PLoS ONE, 2012, 7(8): 42919. doi: 10.1371/journal.pone.0042919 [23] AMINOV R I, CHEE-SANFORD J C, GARRIGUES N, et al. Development, validation, and application of PCR primers for detection of tetracycline efflux genes of gram-negative bacteria[J]. Appl Environ Microbiol, 2002, 68(4): 1786-1793. doi: 10.1128/AEM.68.4.1786-1793.2002 [24] MAO D, LUO Y, MATHIEU J, et al. Persistence of extracellular DNA in river sediment facilitates antibiotic resistance gene propagation[J]. Environ Sci Technol, 2014, 48(1): 71-78. doi: 10.1021/es404280v [25] WANGA L, SU H, HU X, et al. Abundance and removal of antibiotic resistance genes (ARGs) in the rearing environments of intensive shrimp aquaculture in South China[J]. J Environ Sci Health B, 2019, 54(3): 211-218. doi: 10.1080/03601234.2018.1550310 [26] 高盼盼, 罗义, 周启星, 等. 水产养殖环境中抗生素抗性基因 ARGs 的研究及进展[J]. 生毒理学报, 2009, 4(6): 770-779. [27] LUO Y, MAO D, RYSZ M, et al. Trends in antibiotic resistance genes occurrence in the Haihe River, China[J]. Environ Sci Technol, 2010, 44(19): 7220-7225. doi: 10.1021/es100233w [28] 梁惜梅, 聂湘平, 施震. 珠江口典型水产养殖区抗生素抗性基因污染的初步研究[J]. 环境科学, 2013, 34(10): 4073-4082. [29] LIU S, SU H, PAN Y F, et al. Spatial and seasonal variations of antibiotics and antibiotic resistance genes and ecological risks in the coral reef regions adjacent to two typical islands in South China Sea[J]. Mar Pollut Bull, 2020, 158: 111424. doi: 10.1016/j.marpolbul.2020.111424 [30] SHAO S, HU Y, CHENG J, et al. Research progress on distribution, migration, transformation of antibiotics and antibiotic resistance genes (ARGs) in aquatic environment[J]. Crit Rev Biotechnol, 2018, 38(8): 1195-1208. doi: 10.1080/07388551.2018.1471038 [31] AHMED W, ZHANG Q, LOBOS A, et al. Precipitation influences pathogenic bacteria and antibiotic resistance gene abundance in storm drain outfalls in coastal sub-tropical waters[J]. Environ Int, 2018, 116: 308-318. doi: 10.1016/j.envint.2018.04.005 [32] LI A, CHEN L, ZHANG Y, et al. Occurrence and distribution of antibiotic resistance genes in the sediments of drinking water sources, urban rivers, and coastal areas in Zhuhai, China[J]. Environ Sci Pollut Res Int, 2018, 25(26): 26209-26217. doi: 10.1007/s11356-018-2664-0 [33] SU H, HU X, WANG L, et al. Contamination of antibiotic resistance genes (ARGs) in a typical marine aquaculture farm: source tracking of ARGs in reared aquatic organisms[J]. J Environ Sci Health A, 2019, 55(3): 220-229. [34] NOLVAK H, TRUU M, TIIRIK K, et al. Dynamics of antibiotic resistance genes and their relationships with system treatment efficiency in a horizontal subsurface flow constructed wetland[J]. Sci Total Environ, 2013, 461-462: 636-644. doi: 10.1016/j.scitotenv.2013.05.052 [35] HUANG X, ZHENG J, LIU C, et al. Removal of antibiotics and resistance genes from swine wastewater using vertical flow constructed wetlands: effect of hydraulic flow direction and substrate type[J]. Chem Eng J, 2017, 308: 692-699. doi: 10.1016/j.cej.2016.09.110 [36] FANG H, ZHANG Q, NIE X, et al. Occurrence and elimination of antibiotic resistance genes in a long-term operation integrated surface flow constructed wetland[J]. Chemosphere, 2017, 173: 99-106. doi: 10.1016/j.chemosphere.2017.01.027 [37] YUAN Q B, GUO M T, YANG J. Fate of antibiotic resistant bacteria and genes during wastewater chlorination: implication for antibiotic resistance control[J]. PLoS ONE, 2015, 10(3): e0119403. doi: 10.1371/journal.pone.0119403 [38] STANGE C, SIDHU J P S, TOZE S, et al. Comparative removal of antibiotic resistance genes during chlorination, ozonation, and UV treatment[J]. Int J Hyg Environ Health, 2019, 222(3): 541-548. doi: 10.1016/j.ijheh.2019.02.002 [39] WEGRZYN G, WEGRZYN A. Stress responses and replication of plasmids in bacterial cells[J]. Microb Cell Fact, 2002, 1: 2. doi: 10.1186/1475-2859-1-2 [40] SULLIVAN B A, VANCE C C, GENTRY T J, et al. Effects of chlorination and ultraviolet light on environmental tetracycline-resistant bacteria and tet(W) in water[J]. J Environ Chem Eng, 2017, 5(1): 777-784. doi: 10.1016/j.jece.2016.12.052 [41] JEON D, KIM J, SHIN J, et al. Transformation of ranitidine during water chlorination and ozonation: moiety-specific reaction kinetics and elimination efficiency of NDMA formation potential[J]. J Hazard Mater, 2016, 318: 802-809. doi: 10.1016/j.jhazmat.2016.06.039 [42] SHARMA V K, JOHNSON N, CIZMAS L, et al. A review of the influence of treatment strategies on antibiotic resistant bacteria and antibiotic resistance genes[J]. Chemosphere, 2016, 150: 702-714. doi: 10.1016/j.chemosphere.2015.12.084 [43] ZHAO X, SU H, XU W, et al. Removal of antibiotic resistance genes and inactivation of antibiotic-resistant bacteria by oxidative treatments[J]. Sci Total Environ, 2021, 778(2): 146348-146359. -
计量
- 文章访问数: 169
- 被引次数: 0
粤公网安备 44010502001741号
