Stability of Important Veterinary Antibiotics Amoxicillin, Sulfadiazine, and Trimethoprim in Practice-Relevant Model Solutions.

Due to the frequent use of veterinary drugs in animal husbandry, it is important to know their environmental behavior. In this context, little attention has been paid to the stability of the active ingredients in solutions prepared for administration. This is particularly problematic for antibiotics that trigger resistance when administered subtherapeutically. In order to investigate a possible influence of the preparation and storage of veterinary drugs on compound stability, three widely used antibiotics (amoxicillin, sulfadiazine, trimethoprim) were prepared in different model solutions. Depending on their individual stabilities, the incubation period lasted up to 70 days. Samples were analyzed at regular intervals by high-performance liquid chromatography-diode array detection and ultraviolet spectrophotometry. Following official recommendations, the investigations covered various parameters, e.g., pH, buffer substances, influence of light, and temperature. Sulfadiazine was incubated together with trimethoprim at concentrations of 120 mg L-1 and 80 mg L-1 for 70 days. Both compounds proved to be very stable under all experimental conditions and between 92 and 100% of the active ingredients remained. In 0.1% formic acid, a transformation product was found with less than 5% of the parent substance. In contrast, amoxicillin (500 mg L-1) was instable in almost all solutions under investigation. Within 17 days, the concentration of AMO decreased to 72% in ultrapure water. With the exception of a physiological saline solution, the amount of amoxicillin dropped below 10% or even below the detection limit. Thus, a physiological saline solution is best suited for the storage of dissolved amoxicillin for later administration.

Short-Term Test for Toxicogenomic Analysis of Ecotoxic Modes of Action in Lemna minor.

In the environmental risk assessment of substances, toxicity to aquatic plants is evaluated using, among other methods, the 7 dayLemna sp. growth inhibition test following the OECD TG 221. So far, the test is not applicable for short-term screening of toxicity, nor does it allow evaluation of toxic modes of action (MoA). The latter is also complicated by the lack of knowledge of gene functions in the test species. Using ecotoxicogenomics, we developed a time-shortened 3 day assay inLemna minor which allows discrimination of ecotoxic MoA. By examining the changes in gene expression induced by low effect concentrations of the pharmaceutical atorvastatin and the herbicide bentazon at the transcriptome and proteome levels, we were able to identify candidate biomarkers for the respective MoA. We developed a homology-based functional annotation pipeline for the reference genome ofL. minor, which allowed overrepresentation analysis of the gene ontologies affected by both test compounds. Genes affected by atorvastatin mainly influenced lipid synthesis and metabolism, whereas the bentazon-responsive genes were mainly involved in light response. Our approach is therefore less time-consuming but sensitive and allows assessment of MoA in L. minor. Using this shortened assay, investigation of expression changes of the identified candidate biomarkers may allow the development of MoA-specific screening approaches in the future.

Biotransformation of Tetracyclines by Fungi: Challenges and Future Research Perspectives.

Tetracycline antibiotics are used worldwide in human and veterinary medicine. On the basis of low metabolization and through organic fertilizers, tetracyclines enter the environment in a biologically active form. This can have toxic effects on microbial communities and promote the selection of resistant strains. The use of fungi could be a promising approach to deactivate tetracyclines by degradation or derivatization as a result of their particular enzyme endowment. Here, we highlight the current analytical and biotechnological challenges associated with the bioconversion of tetracyclines by fungi and propose research approaches to advance the technology for wastewater and manure treatment.

Innovative Perspectives on Biofilm Interactions in Poultry Drinking Water Systems and Veterinary Antibiotics Used Worldwide.

Prudent use of antibiotics in livestock is widely considered to be important to prevent antibiotic resistance. This study aimed to evaluate the interactions between biofilms and veterinary antibiotics in therapeutic concentrations administrated via drinking water through a standardized experimental setup. In this context, two biofilms formed by pseudomonads (Pseudomonas (P.) aeruginosa or P. fluorescens) and a susceptible Escherichia (E.) coli strain were developed in a nutrient-poor medium on the inner surface of polyvinyl chloride pipe pieces. Subsequently, developing biofilms were exposed to sulfadiazine/trimethoprim (SDZ/TMP) or tylosin A (TYL A) in dosages recommended for application in drinking water for 5 or 7 days, respectively. Various interactions were detected between biofilms and antibiotics. Microbiological examinations revealed that only TYL A reduced the number of bacteria on the surface of the pipes. Additionally, susceptible E. coli survived both antibiotic treatments without observable changes in the minimum inhibitory concentration to 13 relevant antibiotics. Furthermore, as demonstrated by HPLC-UV, the dynamics of SDZ/TMP and TYL A in liquid media differed between the biofilms of both pseudomonads over the exposure period. We conclude that this approach represents an innovative step toward the effective evaluation of safe veterinary antibiotic use.

Comprehensive LC-HRMS metabolomics analyses for the estimation of environmental inputs of altrenogest in pig breeding.

Altrenogest (ALT), a synthetic progestogen, is used in pig farming for estrus synchronization in gilts. Residues of ALT and its metabolites may reach the aquatic environment via the spread of liquid manure and may present a risk for fish and other higher aquatic organisms due to its endocrine disrupting potential. A pilot study was conducted in which spot urine samples from ALT-treated and non-medicated gilts were collected. We applied LC-HRMS analysis to perform targeted analysis of ALT and known metabolites as well as non-targeted metabolomics analyses to find previously unknown metabolites. The targeted investigation showed that glucuronide conjugates of ALT and its photo-isomerization product are main urinary metabolites of ALT in gilts. Furthermore, an unknown isomerization product of ALT was observed at trace level, whereas ALT and ALT sulfate were not found. The chemometric analysis of non-targeted data revealed a clear difference between ALT-treated gilts and control animals. Furthermore, a hydroxylated ALT glucuronide was identified as highly significant in the ALT-treated group. Additional biomarker annotation and pathway mapping revealed changes in the metabolism of ALT-treated animals which can be explained by ALT's hormonal action. This study illustrates the exceptional potential of LC-HRMS and metabolomics for the detection of potentially new environmental contaminants with high biological activity. Further advantages of the method described are the sampling during routine breeding conditions, a relatively small number of animals required and no particular stress for the animals.

Bone toxicity induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and the retinoid system: A causality analysis anchored in osteoblast gene expression and mouse data.

Dioxin exposures impact on bone quality and osteoblast differentiation, as well as retinoic acid metabolism and signaling. In this study we analyzed associations between increased circulating retinol concentrations and altered bone mineral density in a mouse model following oral exposure to 2,3,7,8-tetrachlordibenzo-p-dioxin (TCDD). Additionally, effects of TCDD on differentiation marker genes and genes involved with retinoic acid metabolism were analysed in an osteoblast cell model followed by benchmark dose-response analyses of the gene expression data. Study results show that the increased trabecular and decreased cortical bone mineral density in the mouse model following TCDD exposure are associated with increased circulating retinol concentrations. Also, TCDD disrupted the expression of genes involved in osteoblast differentiation and retinoic acid synthesis, degradation, and nuclear translocation in directions compatible with increasing cellular retinoic acid levels. Further evaluation of the obtained results in relation to previously published data by the use of mode-of-action and weight-of-evidence inspired analytical approaches strengthened the evidence that TCDD-induced bone and retinoid system changes are causally related and compatible with an endocrine disruption mode of action.

Endocrine, metabolic and apical effects of in utero and lactational exposure to non-dioxin-like 2,2',3,4,4',5,5'-heptachlorobiphenyl (PCB 180): A postnatal follow-up study in rats.

PCB 180 is a persistent and abundant non-dioxin-like PCB (NDL-PCB). We determined the developmental toxicity profile of ultrapure PCB 180 in developing offspring following in utero and lactational exposure with the focus on endocrine, metabolic and retinoid system alterations. Pregnant rats were given total doses of 0, 10, 30, 100, 300 or 1000 mg PCB 180/kg bw on gestational days 7-10 by oral gavage, and the offspring were sampled on postnatal days (PND) 7, 35 and 84. Decreased serum testosterone and triiodothyronine concentrations on PND 84, altered liver retinoid levels, increased liver weights and induced 7-pentoxyresorufin O-dealkylase (PROD) activity were the sensitive effects used for margin of exposure (MoE) calculations. Liver weights were increased together with induction of the metabolizing enzymes cytochrome P450 (CYP) 2B1, CYP3A1, and CYP1A1. Less sensitive effects included decreased serum estradiol and increased luteinizing hormone levels in females, decreased prostate and seminal vesicle weight and increased pituitary weight in males, increased cortical bone area and thickness of tibial diaphysis in females and decreased cortical bone mineral density in males. Developmental toxicity profiles were partly different in male and female offspring, males being more sensitive to increased liver weight, PROD induction and decreased thyroxine concentrations. MoE assessment indicated that the 95th percentile of current maternal PCB 180 concentrations do not exceed the estimated tolerable human lipid-based PCB 180 concentration. Although PCB 180 is much less potent than dioxin-like compounds, it shares several toxicological targets suggesting a potential for interactions.

Role of aryl hydrocarbon receptor (AHR) in overall retinoid metabolism: Response comparisons to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) exposure between wild-type and AHR knockout mice.

Young adult wild-type and aryl hydrocarbon receptor knockout (AHRKO) mice of both sexes and the C57BL/6J background were exposed to 10 weekly oral doses of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD; total dose of 200 µg/kg bw) to further characterize the observed impacts of AHR as well as TCDD on the retinoid system. Unexposed AHRKO mice harboured heavier kidneys, lighter livers and lower serum all-trans retinoic acid (ATRA) and retinol (REOH) concentrations than wild-type mice. Results from the present study also point to a role for the murine AHR in the control of circulating REOH and ATRA concentrations. In wild-type mice, TCDD elevated liver weight and reduced thymus weight, and drastically reduced the hepatic concentrations of 9-cis-4-oxo-13,14-dihydro-retinoic acid (CORA) and retinyl palmitate (REPA). In female wild-type mice, TCDD increased the hepatic concentration of ATRA as well as the renal and circulating REOH concentrations. Renal CORA concentrations were substantially diminished in wild-type male mice exclusively following TCDD-exposure, with a similar tendency in serum. In contrast, TCDD did not affect any of these toxicity or retinoid system parameters in AHRKO mice. Finally, a distinct sex difference occurred in kidney concentrations of all the analysed retinoid forms. Together, these results strengthen the evidence of a mandatory role of AHR in TCDD-induced retinoid disruption, and suggest that the previously reported accumulation of several retinoid forms in the liver of AHRKO mice is a line-specific phenomenon. Our data further support participation of AHR in the control of liver and kidney development in mice.

Comprehensive Metabolomics Analysis of Nontargeted LC-HRMS Data Provides Valuable Insights Regarding the Origin of Veterinary Drug Residues.

Liquid chromatography coupled with high resolution mass spectrometry (LC-HRMS) complements standard triple-quadrupole mass spectrometry in veterinary drug residue control. LC-HRMS offers the opportunity for nontargeted screening for metabolites and biomarkers representing metabolic changes. In this work, the feasibility of a nontargeted metabolomics approach based on LC-HRMS data (LC-Q-Orbitrap and LC-Q-TOF) to distinguish between porcine muscle tissue from infected animals and from healthy animals is demonstrated. The differences arise from various compounds associated with metabolic changes in infected animals. Two new biomarker candidates have been identified: tripeptide prolyphenylalanylglycine and a lysophosphatidylcholine derivative. For the first time, a bivariate data analysis procedure is described that may be used to evaluate whether the presence of antibiotic residues points to a therapeutic application or may be the result of a contamination during sampling and/or analysis.

Long-term monitoring of sulfonamides and tetracyclines in manure amended soils and leachate samples - A follow-up study.

Antibiotics can be detected in manure and digestate samples worldwide. As manure is a frequently used fertilizer, antibiotics are found in soil and leachate samples. Only little is known about the long-term fate of antibiotics in the soil environment. One shortcut is the lack of appropriate monitoring studies. Here we present the results of an unequalled soil monitoring study over 18 years from an agricultural field site in Lower Saxony (Germany). Sulfonamides and tetracycline are mainly fixed in the upper soil layer. Contents showed a sharp decrease below sampling depth of 30 cm (plough depth). Sulfaguanidine and sulfamethazine (SMZ) were detected down to 90 cm. Water samples taken below the field site revealed the transfer of sulfonamides into leachate. High variances were observed between sampling points emphasizing the need for sampling strategies for environmental studies. In addition, field lysimeters with defined input of sulfonamides enabled a long-term monitoring and mass balance of antibiotic transfer into leachate over 10 years. SMZ showed the highest mobility with concentrations up to 65 ng L-1. Less than 0.5% of the applied SMZ was transferred into the leachate. Data of lysimeter and field water samples support the theory of a steady state process with a continuous input of sulfonamides such as SMZ into leachate. Soils contaminated with antibiotics can be a long-term source for the input of antibiotic active compounds into deeper soil layers and groundwater.

Ψ-Xenin-6 enhances sitagliptin effectiveness, but does not improve glucose tolerance.

Recent studies have characterised the biological properties and glucose-dependent insulinotropic polypeptide (GIP) potentiating actions of an enzymatically stable, C-terminal hexapeptide fragment of the gut hormone xenin, namely Ψ-xenin-6. Given the primary therapeutic target of clinically approved dipeptidyl peptidase-4 (DPP-4) inhibitor drugs is augmentation of the incretin effect, the present study has assessed the capacity of Ψ-xenin-6 to enhance the antidiabetic efficacy of sitagliptin in high fat fed (HFF) mice. Individual administration of either sitagliptin or Ψ-xenin-6 alone for 18 days resulted in numerous metabolic benefits and positive effects on pancreatic islet architecture. As expected, sitagliptin therapy was associated with elevated circulating GIP and GLP-1 levels, with concurrent Ψ-xenin-6 not elevating these hormones or enhancing DPP-4 inhibitory activity of the drug. However, combined sitagliptin and Ψ-xenin-6 therapy in HFF mice was associated with further notable benefits, beyond that observed with either treatment alone. This included body weight change similar to lean controls, more pronounced and rapid benefits on circulating glucose and insulin as well as additional improvements in attenuating gluconeogenesis. Favourable effects on pancreatic islet architecture and peripheral insulin sensitivity were more apparent with combined therapy. Expression of hepatic genes involved in gluconeogenesis and insulin action were partially, or fully, restored to normal levels by the treatment regimens, with beneficial effects more prominent in the combination treatment group. These data demonstrate that combined treatment with Ψ-xenin-6 and sitagliptin did not alter glucose tolerance but does offer some metabolic advantages, which merit further consideration as a therapeutic option for type 2 diabetes.

Genetic and chemical diversity of the toxic herb Jacobaea vulgaris Gaertn. (syn. Senecio jacobaea L.) in Northern Germany.

Tansy ragwort, Jacobaea vulgaris Gaertn. (syn. Senecio jacobaea L.), is a common Asteraceae in Europe and Asia and known to be an invasive pest in several regions in the world. Recently it is also spreading immensely in native regions like Northern Germany. Pyrrolizidine alkaloids (PAs), which are found in high amounts in Jacobaea vulgaris, are toxic for humans and potentially lethal for grazing animals. In this study we investigated 27 populations of tansy ragwort in Northern Germany for their PA concentration and composition using liquid chromatography coupled to high resolution mass spectrometry. Furthermore, we investigated the genetic structure of selected populations using amplified length polymorphism markers. We detected 98 different PAs in the samples and considerable differences of PA composition between populations. In contrast, PA content of populations did not differ significantly. Genetic (4%) differentiation among populations was low while average genetic diversity was high (0.35). There was no correlation between genetic and geographic distance. Neither genetic markers nor chemical composition revealed any connection to the geographic pattern. As we could not detect any pattern in genetic or chemical diversity, we suggest that the existence of this diversity is a result of a broad interaction with the environment rather than that of evolutionary constraints in the current selection process driving PA composition in J. vulgaris in certain chemotypes.

Degradation and transformation of fluoroquinolones by microorganisms with special emphasis on ciprofloxacin.

Although internationally recognized as the "highest priority critically important antimicrobials," fluoroquinolones are extensively used in both human and veterinary medicine. Poor metabolism and recalcitrance of fluoroquinolones have led to their worldwide presence in municipal wastewaters as well as in manure and, consequently, in several environmental compartments. Being one of the most widely used fluoroquinolones in human medicine and, aside from that, the main metabolite of the veterinary drug enrofloxacin, ciprofloxacin is the most frequently detected fluoroquinolone in effluents of European wastewater treatment plants. Due to serious global concerns about the increasing emergence of bacterial (multi)resistances toward the highly efficient fluoroquinolones, special attention has been paid to their environmental degradation by various microorganisms. This review summarizes research on microbial transformation and degradation of fluoroquinolones with special emphasis on ciprofloxacin, presents an overview of the main ciprofloxacin biotransformation products, and takes a closer look at their biological relevance. Furthermore, own data, experiences, and publications gathered from our recent research in the field are acknowledged.

Determination of adsorbable organically bound fluorine (AOF) and adsorbable organically bound halogens as sum parameters in aqueous environmental samples using combustion ion chromatography (CIC).

Because of their toxicity and biomagnification potential individual perfluoroalkyl and polyfluoroalkyl substances (PFAS) are regularly examined in food and environmental matrices by LC-MS/MS. The combustion ion chromatography (CIC) can be used to determine adsorbable organic fluorine (AOF) in aqueous samples. This report describes the optimization and validation of an automated, robust, cost-efficient and rapid CIC method for the determination of AOF. The analysis of 25 fluorinated organic reference substances was performed with recoveries between 16% and 121%. Water from selected surface waters (n = 74), municipal (n = 116) and industrial wastewaters (n = 33) as well as ground water (n = 93) were analyzed by means of CIC. The AOF values of surface water varied between 2.3 and 24.5 µg/L. The concentrations of AOF in 85% of the wastewater discharges were between 2.0 and 8.5 µg/L, while 15% of the samples were below the limit of quantitation (LOQ = 2 µg/L AOF). In 56% of the ground water samples the values were below the LOQ. In 44% of the surface water samples (n = 41) the values were between 2.0 and 6.1 µg/L AOF. CIC analysis was performed in 22 samples from a chemical company wastewater treatment plant, and 14 individual PFAS were determined by LC-MS/MS. AOF values up to 555 µg/L were found in these samples while the total of the individual PFAS, calculated as fluorine, was 8.8 µg/L. This provides evidence, that CIC covers a huge range of fluoroorganic compounds that are presently not detected by LC-MS/MS. Furthermore, the CIC method allowed the determination of four halogens in 26 surface water samples. This demonstrated that the CIC technique can be used as a powerful screening test to support LC-MS/MS methods, and is also useful to detect organic chlorine, bromine and iodine compounds (AOCl, AOBr and AOI).

Analysis of fluoroquinolones in dusts from intensive livestock farming and the co-occurrence of fluoroquinolone-resistant Escherichia coli.

Fluoroquinolones are important therapeutics in human and veterinary medicine. This study aimed to retrospectively analyse sedimentation dusts from intensive-livestock-farming barns for fluoroquinolones and investigate the association between resistant Escherichia coli and the detected drugs. Sedimentation-dust samples (n = 125) collected (1980-2009) at 14 barns of unknown-treatment status were analysed by HPLC and tandem-mass spectroscopy to detect enrofloxacin, ciprofloxacin, marbofloxacin, and difloxacin. Recent microbiological data were included to investigate the relationship between fluoroquinolone presence and fluoroquinolone-resistant E. coli. Fifty-nine dust samples (47%) from seven barns contained fluoroquinolone residues. Up to three different fluoroquinolones were detected in pig and broiler barns. Fluoroquinolone concentrations ranged from 10-pg/mg to 46-ng/mg dust. Fluoroquinolone-resistant E. coli were isolated from four barns. Of all the dust samples, 22% contained non-susceptible isolates. Non-susceptible isolate presence in the dust was significantly associated (p = 0.0283) with detecting the drugs, while drug detection increased the odds (4-fold) of finding non-susceptible E. coli (odds ratio = 3.9877, 95% CI: 1.2854-12.3712). This retrospective study shows that fluoroquinolone usage leads to dust contamination. We conclude that farmers and animals inhale/swallow fluoroquinolones and fluoroquinolone-resistant bacteria due to drug application. Furthermore, uncontrolled drug emissions via air exhausted from the barns can be assumed.

Biotransformation of ciprofloxacin by Xylaria longipes: structure elucidation and residual antibacterial activity of metabolites.

The impressive ability of the fungus Xylaria longipes to transform the highly persistent fluoroquinolone ciprofloxacin into microbiologically less active degradation products was demonstrated. Fluoroquinolones are used extensively in both human and veterinary medicine. Poor metabolization and high chemical stability of these synthetic antibiotics led to their presence in several environmental compartments. This undesirable behavior may promote the spread of resistance mechanisms due to concomitant exposure to bacteria. Therefore, the biotransformation of ciprofloxacin, one of the most prescribed fluoroquinolones in human medicine, by the ascomycetous soft rot fungus X. longipes was investigated in detail. Submerged cultivation of the fungus allowed for high-yield formation of four biotransformation products. These compounds were subsequently purified by preparative high-performance liquid chromatography. Applying accurate mass spectrometry and nuclear magnetic resonance spectroscopy, desethylene-ciprofloxacin, desethylene-N-acetyl-ciprofloxacin, N-formyl-ciprofloxacin and N-acetyl-ciprofloxacin were unambiguously identified. N-acetylation and N-formylation of the drug led to a 75-88% reduction of the initial antibacterial activity, whereas a breakdown of the piperazine substituent resulted in almost inactive products. These findings suggest an important role in the inactivation and degradation of this and other synthetic compounds in the environment.

Immunochemical Analysis of Paxilline and Ergot Alkaloid Mycotoxins in Grass Seeds and Plants.

Limited availability of toxin standards for lolitrem B and ergovaline impedes routine control of grasses for endophyte toxins. This study aimed at assessing the applicability of an enzyme immunoassay (EIA) for the indole-diterpene mycotoxin paxilline, in combination with a generic EIA for ergot alkaloids, as alternative parameters for screening purposes. Analysis of grass seeds and model pastures of four different grass species showed that both EIAs yielded highly positive results for paxilline and ergot alkaloids in perennial ryegrass seeds. Furthermore, evidence for natural occurrence of paxilline in grass in Germany was obtained. High performance liquid chromatography-tandem mass spectrometry analysis qualitatively confirmed the paxilline EIA results but showed that paxilline analogues 1'-O-acetylpaxilline and 13-desoxypaxilline were the predominant compounds in seeds and grass. In the absence of easily accessible reference standards for specific analysis of some major endophyte toxins, analysis of paxilline and ergot alkaloids by EIA may be suitable substitute parameters. The major advantage of this approach is its ease of use and speed, providing an analytical tool which could enhance routine screening for endophyte toxins in pasture.

Detection of the tremorgenic mycotoxin paxilline and its desoxy analog in ergot of rye and barley: a new class of mycotoxins added to an old problem.

A newly developed enzyme immunoassay (EIA) for the detection of the tremorgenic indole-diterpene alkaloid paxilline (PAX) and closely related analogs was used to analyze ergot sclerotia collected from rye and barley fields. The mean EIA standard curve detection limit was 0.47 ± 0.14 ng/mL; relative cross-reactivity of toxin standard solutions was found for 11-hydroxy-paspaline (terpendole E, 1.1%) but not for lolitrem B or ergot alkaloids. Sclerotia from all fields were positive in the PAX-EIA at concentration levels of 620 ± 200 and 160 ± 37 µg/kg in ergot of rye and 130 ± 47 µg/kg in ergot of barley. Confirmatory analyses of sclerotia by liquid chromatography-tandem mass spectrometric detection identified PAX and its analog 13-desoxypaxilline. To the best of our knowledge, this is the first report on the natural occurrence of tremorgenic indole-diterpene alkaloid mycotoxins in ergot sclerotia from rye and barley. Along with details on the analytical methodology developed in this study, particularly PAX-antibody production, the relevance and implications of these findings for food and feed safety are discussed. Presence or absence of elevated levels of tremorgenic mycotoxins, along with the ergot alkaloids, would help in explaining the difference between the two distinct manifestations of historic ergotism, the convulsive and the gangrenous form. Further method development for paxilline and other tremorgenic mycotoxins in cereals used for food and feed is a prerequisite for a comprehensive risk assessment, which seems to be necessary in light of the findings reported here. Paxilline in ergot of rye.