Astrid Spielmeyer

Elimination patterns of worldwide used sulfonamides and tetracyclines during anaerobic fermentation

Antibiotics such as sulfonamides and tetracyclines are frequently used in veterinary medicine. Due to incomplete absorption in the animal gut and/or unmetabolized excretion, the substances can enter the environment by using manure as soil fertilizer. The anaerobic fermentation process of biogas plants is discussed as potential sink for antibiotic compounds. However, negative impacts of antibiotics on the fermentation process are suspected. The elimination of sulfadiazine, sulfamethazine, tetracycline and chlortetracycline in semi-continuous lab-scale fermenters was investigated. Both biogas production and methane yield were not negatively affected by concentrations up to 38 mg per kg for sulfonamides and 7 mg per kg for tetracyclines. All substances were partly eliminated with elimination rates between 14% and 89%. Both matrix and structure of the target molecule influenced the elimination rate. Chlortetracycline was mainly transformed into iso-chlortetracycline. In all other cases, the elimination pathways remained undiscovered; however, sorption processes seem to have a negligible impact.

The veterinary antibiotic journey: comparing the behaviour of sulfadiazine, sulfamethazine, sulfamethoxazole and tetracycline in cow excrement and two soils

PurposeTo conceptualize the journey that veterinary antibiotics (VAs) follow between the animal stall and the field, two experiments were conducted. (1) The VAs sulfadiazine, sulfamethazine, sulfamethoxazole and tetracycline were mixed into cow excrement; prepared with three dry solid content variations. (2) Cow excrement containing the same VAs was mixed into sandy and clayey saturated soils. The aim was to quantify the solid-liquid partitioning and time-dependent behaviour of VAs at each stage of the journey, to enable mathematical replication of the process in the future.Materials and methodsIn each case, the mixtures were partitioned into their solid and liquid phases and the VA concentration in each was determined using liquid chromatography tandem mass spectrometry. Sorption isotherms (Kd values) and elimination constants (ks: solid form, kl: liquid form) were calculated after various incubation periods.Results and discussionSulfamethoxazole exhibits fast elimination in excrement; environmental contamination is unlikely. Sulfadiazine and sulfamethazine behave similarly in excrement and soils; tetracycline is more sorptive. Small percentages of the sulfadiazine, sulfamethazine and tetracycline masses initially found in excrement may subsequently be transported to environmental compartments in liquid form. However, the majority of these VAs are speculated to accumulate in soil or be transported to surface water systems via soil erosion.ConclusionsThe VA journey has been mathematically conceptualized for the first time and is supported by sorption isotherms and eliminations constants for four commonly detected VAs. Critical findings for sulfadiazine, sulfamethazine and tetracycline are (1) the majority of an initial VA mass resides in excrement liquid; (2) following incorporation into soil, the majority of the same initial VA mass resides in soil solid; (3) VAs found in soil liquid are assumed to be eliminated within a few months; (4) VAs found in soil solid are assumed to persist and accumulate; and (5) VAs are most likely to be transported to surface water systems in solid form (via soil erosion). Due to its rapid elimination in excrement, sulfamethoxazole that stems from veterinary medicine is not assumed to be a major environmental contaminant.

Biotransformation of the Antibiotic Danofloxacin by Xylaria longipes Leads to an Efficient Reduction of Its Antibacterial Activity

Fluoroquinolones are considered as critically important antibiotics. However, they are used in appreciable quantities in veterinary medicine. Liquid manure and feces can contain substantial amounts of unmetabolized antibiotics and, thus, antibiotics can enter the environment if manure is used for soil fertilization. In this study, the microbial biotransformation of the synthetic veterinary fluoroquinolone danofloxacin by the ascomycete Xylaria longipes was investigated. Fungal submerged cultures led to a regioselective and almost quantitative formation of a single metabolite within 3 days. The metabolite was unequivocally identified as danofloxacin N-oxide by high-resolution mass spectrometry and one- and two-dimensional nuclear magnetic resonance spectroscopic techniques. An oxidation of the terminal nitrogen of the substituted piperazine moiety of the substance led to a remarkable reduction of 80% of the initial antibacterial activity. Thus, fungal enzymes involved in the biotransformation process might possess the potential to reduce the entrance of antibiotics via biotransformation of these compounds.

From agricultural fields to surface water systems: the overland transport of veterinary antibiotics

PurposeThe aim of this study was to assess if veterinary antibiotics, which are introduced to agricultural fields via fertilizer, are present in water systems (in both water and sediment) following overland transport via runoff and soil erosion.Materials and methodsThree water and sediment sampling schemes were conducted in Germany between June 2013 and June 2014 to examine the effects of (1) season, (2) heavy rainfall and (3) high veterinary antibiotic usage. The samples were analysed for 15 veterinary antibiotics using liquid chromatography tandem mass spectrometry.Results and discussionAntibiotics were detected in all three schemes in trace quantities. This is the first time that veterinary antibiotics have been detected in German sediment.ConclusionsIn particular, the presence of tetracycline in sediment taken from irrigation ditches in an agricultural area of high veterinary antibiotic usage offers proof that the overland transport of veterinary antibiotics is occurring. Due to the strong sorption of veterinary antibiotics (particularly tetracyclines) to soils, further research into their transport via soil erosion and assessment of their presence in sediment is advised.

Transformation of Sulfonamides and Tetracyclines during Anaerobic Fermentation of Liquid Manure

Liquid manure is frequently used as soil fertilizer due to its high nutrient content. It can also contain residues of pharmaceuticals, such as antibiotics, if farm animals are medicated. The anaerobic fermentation process in biogas plants is discussed as one way to reduce the input of antibiotics into the environment. Therefore, 10 worldwide-applied sulfonamides (sulfachloropyridazine, sulfadiazine, sulfadimethoxine, sulfaguanidine, sulfamerazine, sulfamethazine, sulfamethoxazole, sulfamethoxypyridazine, sulfapyridine, and sulfathiazole) and four frequently used tetracyclines (chlortetracycline, doxycycline, oxytetracycline, and tetracycline) were investigated concerning their elimination pattern during anaerobic fermentation. Batch fermenters with autoclaved and non-autoclaved inoculum were utilized to distinguish between biotic and abiotic elimination pathways. Overall, sulfadimethoxine, sulfamethoxypyridazine, and sulfamethoxazole showed the highest elimination, which was considerably reduced by autoclaving before inoculation. Structure elucidation via nuclear magnetic resonance and different mass spectrometry techniques revealed only minor structural modifications such as O-demethylation and hydrogenation, which did not result in a considerably reduced antimicrobial activity. These results show that, especially, sulfonamides are more persistent than expected. Future studies should deal with the elucidation of relevant process parameters for an enhanced compound degradation.

Efficient Reduction of Antibacterial Activity and Cytotoxicity of Fluoroquinolones by Fungal-Mediated N-Oxidation

Extensive usage of fluoroquinolone antibiotics in livestock results in their occurrence in manure and subsequently in the environment. Fluoroquinolone residues may promote bacterial resistance and are toxic to plants and aquatic organisms. Moreover, fluoroquinolones may enter the food chain through plant uptake, if manure is applied as fertilizer. Thus, the presence of fluoroquinolones in the environment may pose a threat to human and ecological health. In this study, the biotransformation of enrofloxacin, marbofloxacin, and difloxacin by the fungus X. longipes (Xylaria) was investigated. The main metabolites were unequivocally identified as the respective N-oxides by mass spectrometry and nuclear magnetic resonance spectroscopy. Fungal-mediated N-oxidation of fluoroquinolones led to a 77-90% reduction of the initial antibacterial activity. In contrast to their respective parent compounds, N-oxides showed low cytotoxic potential and had a reduced impact on cell proliferation. Thus, biotransformation by X. longipes may represent an effective method for inactivating fluoroquinolones.

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.