Conversion of cephapirin to deacetylcephapirin in milk and tissues of treated animals

Cephapirin is one of six beta-lactam antibiotics approved for use in the treatment of food-producing animals in the United States. When used for treatment of mastitis by intramammary infusion, it is partially converted to a microbiologically active metabolite identified as deacetylcephapirin (DACEP). The degradation was followed in four cows with naturally acquired mastitis which were treated with cephapirin. DACEP persisted longer than the parent compound in the milk. When a calf was treated with cephapirin by intramuscular injection, the compound was almost completely converted to DACEP in tissues. The deacetyl form must be considered in the determination of residues in treated animals.     

土壤中抗生素的环境行为及分布特征研究进展

抗生素被广泛应用于治疗和控制人类及其他动物的细菌感染性疾病,并且可以作为饲料添加剂或生长促进剂用于禽畜养殖。大量抗生素通过有机肥施用、污水灌溉等途径持续进入土壤,导致土壤环境中抗生素种类不断增加、含量逐年增高。抗生素污染会对土壤中微生物、动物、植物等产生直接或间接的影响,诱导耐药菌株及抗性基因的产生,并且抗生素还可以被植物吸收,通过食物链影响人类健康。本文系统介绍了近20年来土壤中抗生素的相关研究,并着重就土壤中抗生素的来源归趋、环境行为以及时空分布情况进行了总结。研究认为土壤中抗生素的环境行为受抗生素种类和土壤性质的强烈影响,并且土壤中抗生素的时空分布与其环境行为和人类活动密切相关。     

Antibiotic residues in animal-derived food

Antibiotics are used extensively in food-producing animals to maintain optimal health and promote growth. The use of these drugs has the potential to leave drug-related residues such as parent drug and metabolites in meat, milk, and eggs. For those drugs that require a withholding period to ensure that residues in the food products are below established tolerances, methods of analysis are required. The majority of approved antibiotics are assayed by microbiological methods. Although these methods are suitable for screening for drug residues, they often lack the specificity and precision required of regulatory methods. Present requirements for regulatory methods have resulted in the submission of physical-chemical methods in support of New Animal Drug Applications for antibiotics.     

Chromatographic methods for determination of macrolide antibiotic residues in tissues and milk of food-producing animals

Tylosin, an antibiotic developed specifically for agricultural use, and erythromycin are the main macrolide antibiotics used in animal production. Two-dimensional thin layer chromatography has been used for detection of tylosin in poultry meat, eggs, and milk and for erythromycin in poultry meat. Detection limits reported are, for tylosin, 0.1 ppm in poultry meat, 0.05 ppm in egg, and 0.01 ppm in milk, and for erythromycin, 0.25 ppm in poultry meat. Liquid chromatography (LC) has also been used for determination of tylosin in milk, blood, and tissues of animals. Samples (milk, blood serum, or tissue homogenates in water or pH 2.2 buffer) were deproteinized with acetonitrile, tylosin was partitioned into methylene chloride, and the extracts were concentrated and dissolved in acetonitrile. Chromatography was done on a reverse phase end-capped C18 column using 0.002-0.005 M ammonium dihydrogen phosphate-acetonitrile-methanol (10 + 60 + 30-5 + 80 + 15). Solvent composition was varied with the type of sample analyzed. The method will detect 0.1 ppm tylosin in tissues and less in milk and blood serum. The LC method was more sensitive than microbiological assays for detection of tylosin in tissues of treated swine; recoveries of tylosin by the LC method were frequently several-fold higher.     

Thin layer chromatographic/bioautographic method for identification of antibiotic residues in animal tissues

An analytical method for the identification of the residues from 14 commonly used antibiotics is presented. The technique is based on selective tissue extraction followed by thin layer chromatography (TLC)/bioautography. Antibiotic residues are extracted from the tissues with methanol and methanol-HCl (98 + 2). The methanol extract is further extracted with chloroform to isolate groups of antibiotics. The extracts are spotted onto TLC plates and developed in suitable solvent systems. Developed plates are placed on set medium seeded with Bacillus subtilis and a bioautograph is produced. The locations of zones of inhibition are used to identify antibiotic residues. Recoveries of antibiotics were quantitative, while the effect of naturally inhibiting components of the matrix was minimized. The sensitivity of the method can be adjusted through minor modifications, which allows its use in routine regulatory analysis.     

Identification of anthocyanins in the liver, eye, and brain of blueberry-fed pigs

Dietary intervention with anthocyanins may confer benefits in brain function, including vision. Research to date indicates that animals have only a limited capacity to absorb anthocyanins, compared to other types of flavonoids. Pigs, which are a suitable model for human digestive absorption, were used to examine the deposition of anthocyanins in tissues including the liver, eye, and brain tissue. Pigs were fed diets supplemented with 0, 1, 2, or 4% w/w blueberries ( Vaccinium corymbosum L. 'Jersey') for 4 weeks. Prior to euthanasia, pigs were fasted for 18-21 h. Although no anthocyanins were detected in the plasma or urine of the fasted animals, intact anthocyanins were detected in all tissues where they were sought. LC-MS/MS results are presented for the relative concentration of 11 intact anthocyanins in the liver, eye, cortex, and cerebellum. The results suggest that anthocyanins can accumulate in tissues, including tissues beyond the blood-brain barrier.     

光催化降解沼液中四环素类抗生素效果及反应动力学研究

该文采用光催化降解途径探究沼液中四环素类抗生素降解的最佳光源、pH值以及光催化对不同初始质量浓度抗生素的降解效果,同时进行不同初始浓度、pH值条件下抗生素光催化降解动力学研究。结果表明:不同光源对四环素类抗生素的降解效果为:高压汞灯紫外消毒灯长弧氙灯无光。高压汞灯催化2 h后,四环素、土霉素、金霉素的降解率分别达到91.68%、85.58%、81.18%。四环素类抗生素的初始质量浓度越低,光催化效果越好。四环素、土霉素、金霉素初始质量浓度为5 mg/L时,其降解率最高可达94.80%、88.35%和95.39%,沼液初始pH值对四环素、金霉素的降解率影响存在显著性差异(P0.05)。当pH值为6时,四环素的降解率最大为96.16%,反应速率常数为1.5971h-1,半衰期为0.355 3 h;当pH值为10时,金霉素的降解率最大为90.47%,反应速率常数为1.084 4 h-1,半衰期为0.338 3 h。沼液初始pH值对土霉素的降解率影响无显著差异(P0.05)。当pH值为10时,3种抗生素的平均降解率最大为89.88%。采用高压汞灯在沼液初始pH值为10时,催化降解5 mg/L四环素类抗生素效果最佳。     

综述中国水环境中抗生素的行为和归趋与采用人工湿地处理

Overuse of antibiotics has become a serious ecological problem worldwide.There is growing concern that antibiotics are losing their effectiveness due to an increased antibiotic resistance in bacteria.During the last twenty years,consumption of antibiotics has increased rapidly in China,which has been cited as one of the world's worst abusers of antibiotics.This review summarizes the current state of antibiotic contamination in China's three major rivers (the Yangtze River,Yellow River,and Pearl River) and illustrates the occurrence and fate of antibiotics in conventional municipal wastewater treatment plants (WWTPs).The analytical data indicate that traditional WWTPs cannot completely remove these concerned pharmaceuticals,as seen in the large difference between the distribution coefficient (Kd) and the uneven removal efficiency of various types of antibiotics.Although constructed wetlands (CWs) offer a potential way to remove these antibiotics from water supplies,knowledge of their mechanisms is limited.There are four main factors affecting the performance of CWs used for the treatment of antibiotics in water supplies,the types and configurations of CWs,hydraulic load rates,substrates,and plants and microorganisms.Further researches focusing on these factors are needed to improve the removal efficiency of antibiotics in CWs.     

粪肥施用土壤抗生素抗性基因来源、转移及影响因素

随着新型抗生素开发速度的不断下降以及抗性基因(Antibiotic resistant genes,ARGs)的快速出现和传播,细菌抗药性和ARGs对公共健康存在威胁,被公认为当前全球亟待解决的难题。虽然土壤本底存在ARGs,但畜禽粪便施用等人类活动加速了ARGs在土壤环境中的扩散和传播。粪肥施入土壤后,其对土壤微生物的抗性选择压力及基因水平转移导致的ARGs扩散转移将持续存在。畜禽粪便中的抗性细菌所携带的ARGs、土壤中抗生素累积导致微生物产生的ARGs和粪肥刺激含有ARGs微生物的繁殖等均为土壤中ARGs的主要来源。土壤中ARGs可以向水体和农作物传移,并随着食物链向动物及人类传播。自然因素(温度、降水、时间和土壤类型)和人为因素(抗生素的含量和种类、粪便种类和处理方式、重金属含量及生物质炭添加)均会影响土壤中ARGs的持久和扩散。目前,粪肥施用土壤中ARGs污染对环境质量及健康的潜在影响并不完全清楚,建议加强模型建立、溯源、生物地理分布、从污染源向环境介质的转移规律、削减措施和机制等方面研究,以有效遏制ARGs在环境中的污染,真正做到畜禽粪便资源化、无害化利用。     

Improving broad specificity hapten recognition with protein engineering

Sulfa antibiotics (sulfonamides) are derivatives of p-aminobenzenesulfonamide that are widely used in veterinary medicine. Foods derived from treated animals may be contaminated with these drugs. However, current immunobased sulfonamide detection methods are unfit for screening of products because they are either too insensitive or specific for a few compounds only. An immunoassay capable of detecting all sulfas in a single reaction would be ideal for screening. For development of a binder capable of binding all sulfas, a protein engineering approach was chosen and the properties of monoclonal antibody 27G3 were improved with mutagenesis followed by selection with phage display. Several different mutant antibodies were isolated. The cross-reaction profile of the best mutant antibody was significantly improved over that of the wild-type antibody: it was capable of binding 9 of the tested 13 sulfonamides within a narrow concentration range and also bound the rest of the sulfas, albeit within a wider concentration range.     

Engineering of a broad specificity antibody for simultaneous detection of 13 sulfonamides at the maximum residue level

Sulfa antibiotics (sulfonamides) are a group of molecules sharing the p-aminobenzenesulfonamide moiety. Sulfonamides are used in veterinary and human medicine. Sometimes, the meat or milk of medicated animals is contaminated with residual sulfonamides. Current analytical methods for sulfonamides are unfit for screening of food, because they are either too laborious, insensitive, or specific for a few sulfa compounds only. A rapid immunoassay for detection of all sulfas in a single reaction would thus be useful. Previously, we used protein engineering to improve the broad specificity of sulfa antibody 27G3. In this study, we improved the best mutant of the previous studies with site-directed mutagenesis. The new mutants recognized different sulfonamides with affinities sufficient for detection of all 13 tested sulfonamides below the MRL level. We furthermore demonstrated the functionality of one mutant in some real sample matrices.     

抗生素在土壤中的吸附行为研究进展

抗生素是人类与动植物疾病防治中被广泛使用的抑菌或杀菌药物,由于其不能完全被机体吸收转化,大部分以原形或代谢物的形式经不同途径进入土壤环境,导致土壤微环境破坏,最终影响人类健康。本文通过文献查阅,综合近几年的研究现状,分析土壤矿物质、有机质、土壤溶液及土壤生物对抗生素吸附作用的机理,探究土壤矿物质、有机质、土壤溶液及土壤生物对抗生素吸附性能的影响,并对抗生素在土壤中的吸附行为进行了展望,以期为土壤中抗生素污染防治提供理论依据。     

Evaluation of a lower tier exposure assessment model for veterinary medicines

Veterinary antibiotics are used in large quantities in the European Union, and one of the key environmental exposure routes is via the application of manure containing excreted antibiotics to arable land as fertilizer. It is a legal requirement to assess the environmental risk of veterinary medicines, and this is done in two stages. A key decision parameter in phase I of these assessments is the predicted environmental concentration (PEC) in soil, and if a trigger value of 100 microg/kg is exceeded, then further phase II studies on the fate, behavior, and effects are carried out. A widely used model to calculate manure and soil PECs is the Uniform Approach. This study evaluated the Uniform Approach in two ways: first, by reviewing existing data, addressing data gaps by performing degradation studies, and then calculating soil and manure PECs for the veterinary antibiotics sulfachloropyridazine, oxytetracycline, and tylosin applied to arable land via liquid pig manure and comparing these data with the results from two field-scale fate studies; second, by collating monitoring data and making a comparison with modeled data. The data comparisons indicated that the Uniform Approach model performed conservatively, with initial PECs being up to 2 orders of magnitude greater than measured environmental concentrations, providing confidence in the use of the model in the risk assessment process, although the assumption of first-order degradation kinetics in the model may underestimate the environmental persistence of veterinary antibiotics.     

Analysis and occurrence of 14 sulfonamide antibacterials and chloramphenicol in honey by solid-phase extraction followed by LC/MS/MS analysis

A method was developed for the analysis of 14 sulfonamide antibiotics and chloramphenicol in honey. These antibiotics have been banned for use in food-producing animals; yet, their residues were found in many samples, illustrating the need for a multiresidue analysis for these antibiotics in honey. The method described here uses an acid hydrolysis step to liberate the sugar-bound sulfonamides followed by a solid-phase extraction to remove potential interferences. Analysis was by liquid chromatography--electrospray ionization--tandem mass spectrometry in negative mode for all 15 analytes. This MRM method generated two structurally significant transitions per compound, and it was designed to conform to U.S. Food and Drug Administration MS confirmation guidelines. It also provides 4-EU identification points. One hundred sixteen samples from 25 countries were analyzed, and 38% were found to contain at least one target antimicrobial. Five different target compounds were found in honey from 13 different countries.     

Thin-layer chromatographic determination of erythromycin and other macrolide antibiotics in livestock products

A method is described for determination of 4 macrolide antibiotics in livestock products. Erythromycin, tylosin, oleandomycin, and spiramycin were extracted from animal tissues, milk, and egg with acetonitrile at pH 8.5. Cleanup was done by adding sodium chloride and dichloromethane, evaporating the organic layer, and subsequent acid/base partitioning. After the antibiotics were separated by thin-layer chromatography (TLC), they were reacted with xanthydrol and could be detected as purple spots down to 0.02 mg/kg without interference by other commonly used therapeutic drugs (23 were tested). Anisaldehyde-sulfuric acid, cerium sulfate-molybdic acid, phosphomolybdic acid, and Dragendorff's reagent proved to be less sensitive as visualizing agents. For quantitation, TLC plates were scanned at 525 nm. Recoveries were between 71 and 96% for erythromycin and tylosin in liver, muscle, and egg at the 0.1-0.5 mg/kg level and 51% for erythromycin in milk at the 0.02 mg/kg level (coefficient of variation = 10-18%). Bioautography with Bacillus subtilis was used to confirm results, in addition to TLC analysis of derivatized antibiotics and liquid chromatography with electrochemical detection. Various derivatization procedures for erythromycin were investigated for improved ultra-violet or fluorescence detection in liquid chromatography.     

Development of a monoclonal antibody-based cELISA for the analysis of sulfadimethoxine. 1. Development and characterization of monoclonal antibodies and molecular modeling studies of antibody recognition

Sulfonamide antibiotics are used to treat a variety of bacterial and protozoan infections in cattle, swine, and poultry. Current residue methods for the analysis of sulfonamides in animal-based food products include bioassays, chromatographic methods (HPLC, GLC), and immunoassays. Most immunoassays have employed highly specific polyclonal antibodies. In this paper, we describe the isolation of monoclonal antibodies against sulfadimethoxine (SDM) that vary in their sensitivities and cross-reactivities against a large number of sulfonamides. The most sensitive monoclonal antibody, designated SDM-18, exhibits an IC(50) value for SDM of 1.53 ppb. Another monoclonal antibody, designated SDM-44, exhibits IC(50) values for six sulfonamides well below the established threshold level of 100 ppb for animal tissues. Molecular modeling studies of the cross-reactive drugs suggest that, depending on the monoclonal antibody, both steric and electronic features govern antibody binding. Due to the diversity of these monoclonal antibodies, it should be possible to design both compound- and class-specific monoclonal antibody-based immunoassays.     

泰乐菌素和土霉素在农业土壤中的消解和运移

长期施用禽畜排泄物可导致抗生素在土壤中的积累, 对环境产生不良影响.为了解进入农田后抗生素的去向及残留动态, 选择2个典型农业土壤, 利用田间小区试验, 研究了田间实际状况下泰乐菌素和土霉素2种抗生素在土壤中的消解与运移行为.研究表明, 抗生素在土壤中的消解和运移与抗生素种类和土壤性质有关.抗生素在砂质土壤(清水砂)中的下移明显高于粘壤土(泥质田), 泰乐菌素在土壤中的垂直迁移强于土霉素.表层土壤中抗生素因降解和下移随时间逐渐下降, 消解速率在试验初期大于后期, 并且土霉素消解速率大于泰乐菌素.砂质土壤中抗生素的消解速率在试验初期明显高于粘壤土, 但至试验后期, 二者渐趋相似.田间条件下测得的抗生素消解速率明显低于实验室条件下, 这可能与抗生素进入田间深层土壤后稳定性增加有关.农田施用抗生素初期产生的径流中含较高浓度的抗生素, 但随时间(10 d之内)很快下降至检测下限以下; 试验初期径流中抗生素浓度为泰乐菌素大于土霉素, 砂质土高于粘壤土.     

Development and validation of an indirect competitive enzyme-linked immunosorbent assay for the screening of tylosin and tilmicosin in muscle, liver, milk, honey and eggs

Incorrect use of tylosin and tilmicosin could result in allergy and select resistance. To monitor the illegal use of these antibiotics in animals, a monoclonal-based indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) has been established. Several haptens were synthesized and conjugated to carrier protein. Female Balb/c mice were inoculated with the four different conjugates to produce monoclonal antibodies according to the schemes of immunization. Aftercell fusion and culture several times, nine hybridoma cell lines were isolated. Only one, 3C4 that has isotype IgG2a, was selected for detailed study. The cross-reactivity of the monoclonal antibody 3C4 to tylosin and tilmicosin was 100% and 51% respectively. The standard curves based on the tylosin and tilmicosin matrix calibration ranged from 2.5 to 40 μg L(-1), with an IC(50) value of 6.1 μg L(-1) and 12.1 μg L(-1), respectively. The limits of detection of the ic-ELISA ranged from 5.1 μg kg(-1) to 13.8 μg kg(-1) in edible animal tissues. The recoveries were 74.1% to 120.7% with less than 18.6% of the coefficient of variation when tylosin and tilmicosin were spiked in various biological matrices with the concentrations of 25.0-200.0 μg kg(-1). Good correlations between the results of the ic-ELISA and high performance liquid chromatography were observed in the incurred tissues. These results suggest that the ic-ELISA is a sensitive, accurate and low-cost method that would be a useful tool for the screening of the residues of tylosin and tilmicosin in muscle, liver, milk, honey and eggs.     

Diplopods as Bioindicators of Soils: Analysis of Midgut of Individuals Maintained in Substract Containing Sewage Sludge

Diplopods feed organic matter in decomposition; however, some environmental factors can promote changes in tissues of these animals. Sewage sludge has been applied for recuperation of physical structure of degraded soil. This work analyzed the influence of the sludge from a city of São Paulo in the midgut of the diplopod Rhinocricus padbergi. After the exposition to sludge, the midgut was prepared for histological and ultra-structural analyses. After 1 week of exposition, there were various glycoprotein globules in the fat body, which appeared, ultrastructurally, little electron dense. In the animals exposed for 2 weeks, there was an intensive renovation of the epithelium with the invasion of regenerative cells, which was observed in the histological and ultra-structural analyses. These data showed that the sludge present various substances that were very hazardous for these animals; more studies were necessary before the application of this in agriculture.     

Pharmaceutical antibiotic compounds in soils – a review

Antibiotics are highly effective, bioactive substances. As a result of their consumption, excretion, and persistence, they are disseminated mostly via excrements and enter the soils and other environmental compartments. Resulting residual concentrations in soils range from a few μg upto g kg^–1 and correspond to those found for pesticides. Numerous antibiotic molecules comprise of a non‐polar core combined with polar functional moieties. Many antibiotics are amphiphilic or amphoteric and ionize. However, physicochemical properties vary widely among compounds from the various structural classes. Existing analytical methods for environmental samples often combine an extraction with acidic buffered solvents and the use of LC‐MS for determination. In soils, adsorption of antibiotics to the organic and mineral exchange sites is mostly due to charge transfer and ion interactions and not to hydrophobic partitioning. Sorption is strongly influenced by the pH of the medium and governs the mobility and transport of the antibiotics. In particular for the strongly adsorbed antibiotics, fast leaching through soils by macropore or preferential transport facilitated by dissolved soil colloids seems to be the major transport process. Antibiotics of numerous classes are photodegraded. However, on soil surfaces this process if of minor influence. Compared to this, biotransformation yields a more effective degradation and inactivation of antibiotics. However, some metabolites still comprise of an antibiotic potency. Degradation of antibiotics is hampered by fixation to the soil matrix; persisting antibiotics were already determined in soils. Effects on soil organisms are very diverse, although all antibiotics are highly bioactive. The absence of effects might in parts be due to a lack of suitable test methods. However, dose and persistence time related effects especially on soil microorganisms are often observed that might cause shifts of the microbial community. Significant effects on soil fauna were only determined for anthelmintics. Due to the antibiotic effect, resistance in soil microorganisms can be provoked by antibiotics. Additionally, the administration of antibiotics mostly causes the formation of resistant microorganisms within the treated body. Hence, resistant microorganisms reach directly the soils with contaminated excrements. When pathogens are resistant or acquire resistance from commensal microorganisms via gene transfer, humans and animals are endangered to suffer from infections that cannot be treated with pharmacotherapy. The uptake into plants even of mobile antibiotics is small. However, effects on plant growth were determined for some species and antibiotics.