Quantification of Veterinary Antibiotics in Pig and Poultry Feces and Liquid Manure as a Non-Invasive Method to Monitor Antibiotic Usage in Livestock by Liquid Chromatography Mass-Spectrometry

Antibiotics are active substances frequently used to treat and prevent diseases in animal husbandry, especially in swine and poultry farms. The use of manure as a fertilizer may lead to the dispersion of antibiotic residue into the environment and consequently the development of antibiotic-resistant bacteria. Most pharmaceutical active ingredients are excreted after administration, in some cases up to 90% of the consumed dose can be found in the feces and/or urine as parent compound. Therefore, due to antibiotic metabolism their residues can be easily detected in manure. This article describes a method for simultaneous analysis of ciprofloxacin, chlortetracycline, doxycycline, enrofloxacin, lincomycin, oxytetracycline, tetracycline, tiamulin, trimethoprim and tylosin in feces, liquid manure and digestate. Antibiotics were extracted from the different matrices with McIlvaine-Na2EDTA buffer solution and the extract was purified by the use two techniques: d-SPE and SPE (Strata-X-CW cartridges) and final eluent was analyzed by LC-MS and LC-MS/MS. The European Commission Decision 2002/657/EC was followed to conduct the validation of the method. Recoveries obtained from spiked pig and poultry feces and liquid manures samples ranged from 63% to 93% depending on analytes. The analysis of 70 samples (feces, liquid manure and digestate) revealed that 18 samples were positive for the presence of doxycycline, oxytetracycline, tetracycline, chlortetracycline, enrofloxacin, tiamulin and lincomycin. The results obtained in the presented study demonstrated that animal feces can be used as a non-invasive method detection antibiotic usage in animal productionThe study was financially supported by the KNOW (Leading National Research Centre) Scientific Consortium “Healthy Animal—Safe Food”, decision of Ministry of Science and Higher Education No. 05-1/KNOW2/2015S

Monitoring the presence of 13 active compounds in surface water collected from rural areas in northwestern Spain

Drug residues are considered environmental contaminants, and their occurrence has recently become a matter of concern. Analytical methods and monitoring systems are therefore required to control the continuous input of these drug residues into the environment. This article presents a suitable HPLC-ESI-MS/MS method for the simultaneous extraction, detection and quantification of residues of 13 drugs (antimicrobials, glucocorticosteroids, anti-inflammatories, anti-hypertensives, anti-cancer drugs and triphenylmethane dyes) in surface water. A monitoring study with 549 water samples was carried out in northwestern Spain to detect the presence of drug residues over two sampling periods during 2010, 2011 and 2012. Samples were collected from rural areas with and without farming activity and from urban areas. The 13 analytes were detected, and 18% of the samples collected showed positive results for the presence of at least one analyte. More collection sites were located in rural areas than in urban areas. However, more positive samples with higher concentrations and a larger number of analytes were detected in samples collected from sites located after the discharge of a WWTP. Results indicated that the WWTPs seems to act as a concentration point. Positive samples were also detected at a site located near a drinking water treatment plantThe authors wish to thank the Fondo Europeo Agrícola de Desarrollo Rural (FEADER) and the Consellería de Medio Rural for funding this study through the project FMR331A, and the staff of the Cactus of the University of Santiago de Compostela for their help in the analysis of the samplesS

Presencia y resistencia a antimicrobianos de Escherichia coli aislados a partir de alimentos en el estado de Hidalgo (México)

The presence of Escherichia coli in foods taken from the grocery stores and the supermarkets in Hidalgo State (Mexico) was determined for 73 samples of poultry meat, 60 samples of pork, 86 samples of beef, and 66 samples of vegetables. A total of 352 E. coli strains were isolated, identified, and analyzed by an agar disk diffusion assay for their resistance to 10 antimicrobials. Poultry meat and vegetables taken from groceries showed significantly higher counts (P = 0.0002 and P = 0.0461, respectively) when compared with the samples taken from supermarkets. Compared with the isolates recovered from other foods, E. coli isolated from chicken meat had higher levels of antimicrobial resistance against all antimicrobials tested, with the exceptions of nitrofurantoin resistance of isolates from pork and streptomycin resistance in isolates from pork and beef. In addition, the E. coli isolates from samples taken from the groceries showed higher resistance rates than the isolates from samples taken from the supermarkets for the cases of pork isolates resistance to ampicillin (P = 0.0497), chloramphenicol (P = 0.0075), doxycycline (P = 0.002), and streptomycin (P = 0.0094) and beef isolates resistance against ampicillin (P = 0.0048), streptomycin (P = 0.002), and sulfisoxazole (P = 0.003). The present study revealed that the observed resistance rates correlated well with those reported in the national surveillance programmes of developed countries, with the exception of isolates from chicken meat, which have higher resistance rates. Also, from a microbiological safety point of view, samples taken from supermarkets were in a much better conditions than those obtained from the groceriesLa presencia de Escherichia coli fue investigada en 73 muestras de carne de pollo, 60 muestras de carne de cerdo, 86 muestras de carne de vacuno y 66 muestras de alimentos vegetales muestreados en pequeños ultramarinos y supermercados en el estado de Hidalgo (México). Un total de 352 aislamientos de E. coli fueron seleccionados, identificados y analizados mediante el método de difusión en disco para determinar su resistencia a 10 antimicrobianos. Las muestras de pollo y vegetales obtenidas en tiendas de alimentación mostraron recuentos de E. coli significativamente mayores (P = 0,0002 y P = 0,0461 respectivamente) que las obtenidas en supermercados. Comparados con los procedentes de los restantes alimentos, los E. coli obtenidos a partir de carne de pollo mostraron un mayor grado de resistencia a todos los antimicrobianos estudiados, excepto en lo referente a la nitrofurantoína en el caso de los aislados a partir de carne de cerdo y la estreptomicina respecto de los aislamientos de carne de cerdo y vacuno. Adicionalmente, en algunos casos, los E. coli obtenidos a partir de alimentos muestreados en pequeños ultramarinos mostraron mayores tasas de resistencia que los procedentes de alimentos muestreados en supermercados. Esto ocurrió en los aislamientos procedentes de carne de cerdo para la ampicilina (P = 0,0497), cloranfenicol (P = 0,0075), doxiciclina (P = 0,002) y estreptomicina (P = 0,0094), y en el caso de la carne de vacuno para la ampicilina (P = 0,0048), estreptomicina (P = 0,002) y sulfizoxazol (P = 0,003). El presente estudio demuestra que las tasas de resistencia observadas son compatibles con las publicadas en los programas nacionales de vigilancia de los países desarrollados, con la excepción de los aislamientos procedentes de carne de pollo, en los cuales las tasas de resistencia tienden a ser mayores. Además, desde el punto de vista de la seguridad microbiológica, las muestras obtenidas en supermercados mostraron condiciones significativamente mejores que las obtenidas en tiendas de alimentaciónThe authors are thankful for financial support from Dirección Xeral de Investigación, Desenvolvemento e Innovación (Xunta de Galicia) (grant IN843A2007/42), and CONACYT (Project CB-2006–61310)S

A Confirmatory Method Based on HPLC-MS/MS for the Detection and Quantification of Residue of Tetracyclines in Nonmedicated Feed

The Commission Regulation 574/2011/EC set up maximum levels of coccidiostats and histomonostats in nonmedicated feed as a consequence of carry-over during manufacturing. Carry-over takes place from medicated to nonmedicated feed during feed production. Similar contamination could also occur for other pharmaceuticals such as tetracyclines, a group of antibiotics commonly employed in food production animal. The objective of this work is to present a simple and fast method for the simultaneous detection of four tetracyclines (chlortetracycline, doxycycline, oxytetracycline, and tetracycline) in nontarget feed at a g/kg level. Validation of the method was performed according to the guideline included in the Commission Decision 2002/657/EC for official method. The validated method was successfully applied to 50 feed samples collected from different milk farms and 25 samples obtained from feed manufacturers. While oxytetracycline was the tetracycline most frequently detected, chlortetracycline was the analyte measured at the highest concentration 15.14 mg/Kg. From 75 nonmedicated feed analysed 15% resulted to be positive for the presence of one tetracyclineThe authors wish to thank the Consellería de Cultura, Educación e Ordenación Universitaria, Xunta de Galicia (Project EM 2012/153), for financial supportS

Caracterización molecular de bacterias ácido-lácticas aisladas a partir de carne de ternera envasada al vacío de modo tradicional y mediante un sistema avanzado

A total of 91 lactic acid bacteria (LAB) were isolated from 50 beef samples, 25 packaged by traditional vacuum packaging and 25 packaged using advanced vacuum skin packaging. The isolated LAB were identified using 16S rRNA sequencing, whereas randomly amplified polymorphic DNA (RAPD-PCR) and cluster analysis were used for typing the LABs. Ten different species of LAB were identified and assigned to the following species: Enterococcus gilvus (22 isolates), Enterococcus faecium (9 isolates), Enterococcus casseliflavus (8 isolates), Enterococcus faecalis (4 isolates), Enterococcus malodoratus (3 isolates), Enterococcus devriessei (3 isolates), Lactobacillus sakei (15 isolates), Carnobacterium divergens (12 isolates), Carnobacterium maltaromaticum (5 isolates), and Leuconostoc mesenteroides (8 isolates). The RAPD profile bands observed were not significantly discriminatory, with exceptions of E. casseliflavus, E. faecalis, and E. faecium, which suggests that the type of packaging system used had no specific effect on the selection of most microbiota in the meat after packagingUn total de 91 bacterias ácido-lácticas (LAB) fueron aisladas a partir de 50 muestras de carne de ternera, de las cuales 25 fueron envasadas al vacío de modo tradicional y 25 fueron envasadas mediante un sistema de vacío avanzado. Los aislamientos fueron identificados a través de la secuenciación del ADN ribosomal 16S, mientras que la caracterización de las LAB se realizó mediante la amplificación al azar del ADN polimórfico (RAPD-PCR) y su posterior análisis cluster. Fueron identificadas cepas pertenecientes a diez especies bacterianas diferentes, en concreto: Enterococcus gilvus (22 aislamientos), E. faecium (9 aislamientos), E. casseliflavus (8 aislamientos), E. faecalis (4 aislamientos), E. malodoratus (3 aislamientos), E. devriessei (3 aislamientos), Lactobacillus sakei (15 aislamientos), Carnobacterium divergens (12 aislamientos), C. maltaromaticum (5 aislamientos) y Leuconostoc mesenteroides (8 aislamientos). Los perfiles de bandas de ADN obtenidas no revelaron diferencias significativas, con las excepciones de E. casseliflavus, E. faecalis y E. faecium, sugiriendo que el tipo de envasado no tiene un efecto específico en la selección de la mayor parte de la microbiota presente en la carne envasadaS

Determination of the Presence of Three Antimicrobials in Surface Water Collected from Urban and Rural Areas

Due to the continuous release of antimicrobials into the environment, the aim of this study was to compare the frequency of detection of sulfamethazine, sulfamethoxypyridazine and trimethoprim in surface water collected from urban and rural areas in Northwestern Spain. A monitoring study was conducted with 314 river water samples analyzed by high-performance liquid chromatography coupled to tandem mass spectrometry. The results indicated that 37% of the samples contained residues of at least one of the investigated antimicrobials, and every sampling site yielded positive samples. At sites located near the discharge points of wastewater treatment plants and near the collection point of a drinking-water treatment plant, more than 6% of the samples were positive for the presence of antimicrobial residuesThe authors wish to thank the Fondo Europeo Agrícola de Desarrollo Rural (FEADER) and the Consellería de Medio Rural for funding this study through the Project FMR331AS

Analysis of Tetracyclines in Medicated Feed for Food Animal Production by HPLC-MS/MS

The use of medicated feed is a common practice in animal food production to improve animal health. Tetracyclines and β-Lactams are the groups that are most frequently added to this type of feed. The measurement of the concentration of the analytes in these types of samples is sometimes due to the matrix characteristic, and manufacturers are demanding fast, precise and reproducible methods. A rapid confirmatory method based on a simple extraction protocol using acidified methanol and followed by high performance liquid chromatography coupled to a tandem mass spectrometer for the quantification of four tetracyclines in feed is presented. Validation was performed following the guidelines of Decision 2002/657/EC. Results indicated that the four tetracyclines can be identified and quantified in a concentration range between 50 and 500 mg/kg with recoveries between 84% and 109% and RSD for precision under reproducible conditions between 12% and 16%. Satisfactory results were also obtained with interlaboratory studies and by comparing the method with an HPLC-Fluorescent methodThe authors wish to thank the Consellería de Cultura, Educación e Ordenacion Universitaria, Xunta de Galicia (project EM 2012/153) for financial supportS

Efficacy of Different Waste and By-Products from Forest and Food Industries in the Removal/Retention of the Antibiotic Cefuroxime

Environmental pollution due to antibiotics is a serious problem. In this work, the adsorption and desorption of the antibiotic cefuroxime (CFX) were studied in four by-products/residues from the forestry and food industries. For this, batch-type experiments were carried out, adding increasing concentrations of CFX (from 0 to 50 µmol L−1) to 0.5 g of adsorbent. The materials with a pH higher than 9 (mussel shell and wood ash) were those that presented the highest adsorption percentages, from 71.2% (23.1 µmol kg−1) to 98.6% (928.0 µmol kg−1). For the rest of the adsorbents, the adsorption was also around 100% when the lowest concentrations of CFX were added, but the percentage dropped sharply when the highest dose of the antibiotic was incorporated. Adsorption data fitted well to the Langmuir and Freundlich models, with R2 greater than 0.9. Regarding desorption, the materials that presented the lowest values when the highest concentration of CFX was added were wood ash (0%) and mussel shell (2.1%), while pine bark and eucalyptus leaves presented the highest desorption (26.6% and 28.6%, respectively). Therefore, wood ash and mussel shell could be considered adsorbents with a high potential to be used in problems of environmental contamination by CFXThis research was funded by SPANISH MINISTRY OF SCIENCE, INNOVATION AND UNIVERSITIES, grant numbers RTI2018-099574-B-C21 and RTI2018-099574-B-C22S

Retention of the Antibiotic Cefuroxime onto Agricultural and Forest Soils

Antibiotics in wastewater, sewage sludge, manures, and slurries constitute a risk for the environment when spread on soils. This work studies the adsorption and desorption of the antibiotic cefuroxime (CFX) in 23 agricultural and forest soils, using batch-type experiments. Our results show that the adsorption values were between 40.75 and 99.57% in the agricultural soils, while the range was lower (from 74.57 to 93.46%) in forest soils. Among the Freundlich, Langmuir, and Linear models, the Freundlich equation shows the best fit for the adsorption results. In addition, agricultural soils with higher pH are the ones that present the highest adsorption. Further confirmation of the influence of pH on adsorption is given by the fact that Freundlich’s KF parameter and the Linear model Kd parameter shows a positive correlation with pH and with the exchangeable Ca and Mg values, which are known to affect the charges of the soil colloids and the formation of cationic bridges between adsorbents and adsorbate. In addition, Freundlich’s n parameter shows a positive and significant correlation with the organic matter content, related to the high adsorption taking place on forest soils despite their pH < 5. Regarding desorption, in most cases, it is lower than 1%, which indicates that CFX is adsorbed in a rather irreversible way onto these soils. Overall, these results can be considered relevant regarding their potential impact on environmental quality and public healthThis research was funded by Spanish Ministry of Science, Innovation and Universities, grant numbers RTI2018-099574-B-C21 and RTI2018-099574-B-C22S