畜禽粪便中四环类抗生素的残留和环境行为

我国畜禽养殖业很发达,畜禽粪便中残留的四环素类抗生素的污染对生态环境构成了巨大的潜在威胁。针对这种状况,本文总结了我国四环素类抗生素在畜禽粪便中的残留状况及其进入环境的途径,并概述了其在环境中的迁移和降解状况。     

Acquisition and Spread of Antimicrobial Resistance: A tet(X) Case Study

Understanding the mechanisms leading to the rise and dissemination of antimicrobial resistance (AMR) is crucially important for the preservation of power of antimicrobials and controlling infectious diseases. Measures to monitor and detect AMR, however, have been significantly delayed and introduced much later after the beginning of industrial production and consumption of antimicrobials. However, monitoring and detection of AMR is largely focused on bacterial pathogens, thus missing multiple key events which take place before the emergence and spread of AMR among the pathogens. In this regard, careful analysis of AMR development towards recently introduced antimicrobials may serve as a valuable example for the better understanding of mechanisms driving AMR evolution. Here, the example of evolution of tet(X), which confers resistance to the next-generation tetracyclines, is summarised and discussed. Initial mechanisms of resistance to these antimicrobials among pathogens were mostly via chromosomal mutations leading to the overexpression of efflux pumps. High-level resistance was achieved only after the acquisition of flavin-dependent monooxygenase-encoding genes from the environmental microbiota. These genes confer resistance to all tetracyclines, including the next-generation tetracyclines, and thus were termed tet(X). ISCR2 and IS26, as well as a variety of conjugative and mobilizable plasmids of different incompatibility groups, played an essential role in the acquisition of tet(X) genes from natural reservoirs and in further dissemination among bacterial commensals and pathogens. This process, which took place within the last decade, demonstrates how rapidly AMR evolution may progress, taking away some drugs of last resort from our arsenal.     

超高效液相色谱-电喷雾串联质谱法同时测定生菜中四环素类抗生素及其代谢产物

目的建立超高效液相色谱-电喷雾串联质谱法(ultra performance liquid chromatography-tandem mass spectrometry,UPLC-MS/MS)测定生菜中9种四环素类抗生素及其代谢产物的残留分析方法。方法生菜样品采用EDTA-Mcllvaine缓冲液与乙腈混合溶液提取,HLB固相萃取小柱净化,色谱分离选择ACQUITY UPLC BEH C18色谱柱,以乙腈和0.2%甲酸水溶液为流动相进行梯度洗脱,得到的分离效果较好。质谱采用电喷雾正离子电离、多反应监测模式检测。结果在0.01~0.5 mg/L范围内线性相关系数r2均大于0.99。仪器检出限为1.085~15.780μg/kg,方法的定量限为3.617~42.526μg/kg。在4个添加水平0.01、0.05、0.1和0.5 mg/kg下,除脱水四环素和差向脱水四环素(其回收率40%)外,其余四环素类抗生素及代谢产物在蔬菜中的添加回收率在为63.4%~101.3%,相对标准偏差为2.52%~12.56%。结论该方法灵敏度高,净化效果好,可以用于蔬菜中四环素类抗生素及其代谢产物的残留分析。     

基于单链DNA模板合成铜纳米簇的四环素无标记检测

以聚T单链DNA为模板合成的铜纳米簇作为荧光探针,建立了一种快速、非标记型的荧光生物传感检测新方法,并用于实际样品中四环素的检测。结果表明,该方法用于检测四环素残留的检出限为0.01μmol/L,用于实际样品四环素残留的测定低限为48μg/kg,而且无需要任何荧光染料标记或复杂的DNA序列设计,所有过程均在室温下10 min内完成。将该方法应用于牛奶、鸡肉样品中四环素含量的测定,相对标准偏差≤4.01%,加标回收率为97.67%~110.23%。     

超高效液相色谱-串联质谱法同时测定鳗鱼中32种兽药残留

目的 建立超高效液相色谱-串联质谱法(ultra performance liquid chromatography-tandem mass spectrometry, UPLC-MS/MS)同时测定鳗鱼中13种磺胺类、15种喹诺酮类和4种四环素类兽药残留的分析方法。方法 鳗鱼试样采用乙二胺四乙酸二钠(ethylenediaminetetraacetic acid disodium salt, Na2EDTA)-Mcllvaine提取液进行提取, HLB固相萃取柱净化, Waters ACQUITY UPLC BEH C18色谱柱分离, 以0.1%甲酸乙腈和0.1%甲酸水溶液作为流动相进行梯度洗脱; 电喷雾正离子模式下, 以多反应监测(multiple reaction monitoring, MRM)方式采集数据进行定性与定量分析, 外标法定量。结果 磺胺类、喹诺酮类及四环素类药物分别在2~50、2~50、5~100 μg/kg浓度范围内线性相关性良好, 相关系数均大于0.99; 不同加标水平的平均回收率为82.6%~110.2%, 相对标准偏差(relative standard deviations, RSDs)为3.2%~13.9%, 检出限(limits of detection, LODs)和定量限(limits of quantification, LOQs)分别为0.3~2.0 μg/kg和1.0~5.0 μg/kg。结论 本方法快速简便、灵敏可靠, 适合大批量鳗鱼中同时快速对磺胺类、喹诺酮类及四环素类兽药残留进行定性和定量检测。     

Development and validation of multi-residue analysis for tetracycline antibiotics in feed by high performance liquid chromatography coupled to mass spectrometry

ABSTRACT

A new multi-residue method for the analysis of the tetracycline antibiotics oxytetracycline, tetracycline, chlortetracycline, and doxycycline was developed and validated for animal feed. After extraction with 0.1M Na₂EDTA-McIlvaine buffer (pH 4), the samples were centrifuged, purified by SPE (Strata-X-CW cartridges) and analysed by HPLC-MS. Validation of the method was performed according to the guidelines indicated in European Commission Decision 2002/657/EC. The procedure was validated by spiking feed samples at three different levels (300, 1000, and 5000 µg/kg). Average recoveries for tetracyclines were in the range 79.70–98.8%, with RSD for repeatability and reproducibility in the ranges of 5.0–9.5% and 6.5–11.0%, respectively. The method was successfully validated and proved to be efficient, precise, and useful for quantification of tetracyclines in animal feed. The validated method was successfully applied to 65 suspect feed samples collected from different regions of Poland in 2015–16 and obtained from farms and feed manufactures. Of these 65 purportedly non-medicated feeds, eight (12.3%) were positive for the presence of doxycycline or chlortetracycline.     

城市污水厂中四环素类抗生素分布特性研究

使用HPLC-MS/MS检测江苏某城市污水厂A中3种四环素抗生素分布状况,并利用质量平衡分析其在污水厂中的归趋及去除特性。结果显示进水中3种四环素含量分别是:四环素336 ng·L-1,土霉素202 ng·L-1和金霉素96 ng·L-1。而出水中只检测到四环素含量为130 ng·L-1,土霉素和金霉素都未检出。结合实测数据,通过质量平衡计算可知,污水厂A中污泥吸附对3种四环素的降解率分别为四环素28.8%,土霉素38.0%和金霉素34.9%。生物降解对3种四环素的去除率分别为四环素为32.5%,土霉素为62.0%,金霉素为65.1%。     

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Screening zum Antibiotika-Transfer aus dem Boden in Getreide in Regionen Nordrhein-Westfalens mit großen Viehbeständen

Zusammenfassung:  In einem vorangegangenen Forschungsprojekt wurde ein Transfer von Antibiotika aus güllebeaufschlagtem Boden in den als Nahrungs- und Futtermittel genutzten Teil des Getreides, das Korn, nachgewiesen. Unklar blieb jedoch, ob dieser Transfer auch in der Praxis der konventionellen Landwirtschaft von Bedeutung ist, da in der Regel die Antibiotikagehalte in Güllen aus der landwirtschaftlichen Praxis geringer sind als die Gehalte, die unter den Bedingungen des o. a. Projektes erreicht wurden. Mit Unterstützung der Landwirtschaftskammern Nordrhein-Westfalen und Niedersachsen wurden in den Jahren 2005 und 2006 in den Kreisen Borken, Coesfeld und Warendorf (nur 2005) sowie der Grafschaft Bentheim (nur 2006) aus der laufenden Ernte sowohl Bodenproben als auch Weizen-, Gerste- und Triticaleproben gezogen. Die Analyse dieser Proben, die Gebieten mit hohem Viehbesatz entstammten, führte zu sicher identifizierten positiven Befunden für Tetracyclin (TC), Chlortetracyclin (CTC), Isochlortetracyclin (iso-CTC), Doxycyclin (DC) und Demeclocyclin (DMC). Die Gehalte lagen vielfach unter der jeweiligen Bestimmungsgrenze (~8–18 μg/kg Frischgewicht (FG)), in seltenen F?llen im Bereich zwischen ~30 (iso-CTC, DC), ~60 (DMC, CTC) und maximal ~95 μmg/kg FG (iso-CTC). Ein direkter Zusammenhang zwischen der Medikamentierung der jeweiligen Viehbest?nde mit Tetracyclinen und den nachgewiesenen Tetracyclin-Gehalten in Boden und Korn ist nicht erkennbar. Die Antibiotikagehalte in Boden und Getreide korrelieren ebenfalls nur teilweise. Dieses k?nnte auf eine Remobilisierung zuvor an Bodenpartikel gebundener Antibiotika hinweisen. Im Getreide der Erntezeit aus dem Jahr 2006, das durch extreme Trockenheit w?hrend der Wachstumsperiode gekennzeichnet war, wurden keine positiven Tetracyclin-Befunde festgestellt. Ungekl?rt bleiben sowohl die relativ hohen Gehalte an DMC im Winterweizen der Erntezeit des Jahres 2005 aus dem Kreis Borken als auch die Befunde an DC in drei Proben aus Niedersachsen der Ernte aus dem Jahr 2006. In der vorangegangenen Studie war ein Transfer von CTC und iso-CTC in die Getreidepflanze auf den Versuchsfl?chen nachweisbar, die zweimal organisch (mit definiert Antibiotika-haltiger Gülle) gedüngt worden waren. Die Ergebnisse der vorliegenden Studie geben Hinweise, dass auch in der landwirtschaftlichen Praxis ein Antibiotikatransfer bis in das Getreidekorn stattfinden kann. Zweifellos wird ein hoher und langfristiger Einsatz von Tetracyclinen der dominierende Faktor sein. Esbesteht nach der jetzigen, noch sehr schmalen Datenbasis aber kein Grund zur Annahme, dass in Gebieten mit hohem Viehbesatz mit einer fl?chendeckenden Belastung des Getreides durch Antibiotika aus Gülle zu rechnen ist.

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Summary:  Screening of antibiotic transfer from soil into cereals in areas with high livestock concentration in Northrhine Westphalia (Germany) In a former investigation transfer of antibiotics from slurry fertilised soil into the grain of winter wheat was demonstrated. Unclear remained, however, whether such a transfer also occurs in commercial agriculture as antibiotic concentrations are usually lower in those slurries than in the one used in the study mentioned above. Supported by the chambers of agriculture in Northrhine Westphalia and Lower Saxony, grain and soil samples were collected in the years 2005 and 2006 in the districts of Borken, Coesfeld and Warendorf (only in 2005) as well as Grafschaft Bentheim (only in 2006). Analyses of these grain samples from areas with high livestock concentration led to verified positive findings of tetracycline, chlortetracycline, iso-chlortetracycline, doxycycline and demeclocycline. Concentrations were usually below detection limit (~8–18 μg/kg fresh matter), in rare cases ranged between ~30 (iso-chlortetracycline, doxycycline), ~60 (demeclocycline, chlortetracycline) and at most ~95 μg/kg fresh matter (iso-CTC). A close correlation between livestock medication with tetracycline and detected tetracycline concentrations in soil and grain was not found. Likewise, antibiotic content in soil and grain did not always correspond. This might indicate remobilisation of antibiotics previously bound to soil particles. In grain from harvest of 2006 no positive tetracycline concentrations were detected (areas of Borken and Coesfeld: winter wheat, barley and triticale). Ambiguous are the relative high concentrations of demeclocycline in winter wheat of harvest in 2005 in the area of Borken as well as the findings of doxycline (DC) in the samples from Lower Saxony in 2006. In the former study we demonstrated a transfer of CTC and iso-CTC into the cereal on those test fields that were fertilised twice with antibiotic containing slurry in defined concentrations. The results of this study give evidence that such a transfer can occur in commercial agriculture as well. Beyond doubt a high and long term application of tetracycline will be the dominating factor. However, there is no evidence that even in areas with high livestock concentration an area wide antibiotic load has to be anticipated.

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Eingegangen: 3. Dezember 2007; Angenommen: 23. Januar 2008     

超高效液相色谱-串联质谱法分析动物源性食品中4种四环素类药物残留

目的建立超高效液相色谱-质谱联用法(ultra performance liquid chromatography-tandem mass spectrometry, UPLC-MS/MS)检测动物源性食品中四环素类药物(四环素、土霉素、金霉素、强力霉素)残留量的方法。方法样品经Na_2EDTA-Mcllvaine缓冲液(pH=4.0)提取,HLB固相萃取柱净化。采用超高效液相色谱-串联三重四极杆质谱法多反应监测(multiple reaction monitoring, MRM)模式采集,外标法进行定性和定量检测。色谱柱为Waters ACQUITY BEH C_(18)(100mm×2.1mm,1.7μm),以20 mmol/L草酸溶液-乙腈为流动相梯度洗脱,流速0.3 mL/min。结果四环素、土霉素、金霉素、强力霉素在5～200μg/L浓度范围内均具有良好的线性关系(r 0.995),检出限均为2.5μg/kg。四环素、土霉素、金霉素、强力霉素的平均回收率分别为100.8%～105.7%、93.1%～107.2%、94.1%～96.2%和92.7%～99.3%,相对标准偏差(relative standard deviation,RSD)8%。结论本方法选择性强,灵敏度高,快速,重现性好,可用于动物源性食品中四环素类药物残留的快速测定。