Developing a microbiological growth inhibition screening assay for the detection of 27 veterinary drugs from 13 different classes in animal feedingstuffs

Abstract

Many regulations prohibit using veterinary drugs in feedingstuffs to protect consumers and animals alike. Within this investigation we developed a simple, cost-efficient primary screening method for detecting antibiotics and coccidiostats in animal feeds. Thirty-two veterinary drugs were originally considered. Following matrix-free testing to optimise detection, an assay based on matrix extraction with methanol/acetonitrile/phosphate buffer followed by inoculation and diffusion in agar plates was developed. Final validation was performed with 14 representative drugs (one per drug class) and four bacteria (Escherichia coli ATCC11303 and ATCC27166, Staphylococcus aureus ATCC6538P, Micrococcus luteus ATCC9341) in bovine, lamb and swine fodder, measuring growth inhibition zones. Of the original drugs tested, 27 remained detectable in feed matrices at or below 20 mg kg⁻¹. Of the 14 validated representatives, two had estimated minimum detectable concentrations of 10–11 mg kg⁻¹, others of 5 mg kg⁻¹ or lower, an earlier minimum European Union inclusion rate for many veterinary drugs. No significant matrix effect on inhibition zones was detected. Per cent wrong negative deviations ranged from 0% (nine of 14 compounds) to 20–27% (two of 14), while inter-day precision based on inhibition zones had relative standard deviations (RSDs) of 6–109% (mean of 40%). When setting a 1 mm inhibition zone, the maximum observed for negative controls, as a cut-off level, no false-positives were found. While not all targeted antibiotics were detectable in complex matrices, the majority of veterinary drugs were detected with reasonable sensitivity, indicating that this method could be suitable for screening feedingstuffs prior to further confirmatory investigation of positive findings such as by LC-MS/MS.

Graphical abstract

Keywords:

• antibiotics
• coccidiostats
• animal feeds
• growth inhibition
• matrix
• multi-method

Acknowledgements

The authors extend their appreciation to Boris Untereiner and Servane Contal for matrix extractions and microbiological examinations, respectively. Many thanks to Dr Frank Schaul and Frederic Navette for the donation of the test matrices; and to Danielle Ruckert for her valuable ideas on the experiments. T. B. planned the general experiments and wrote the manuscript; T. P. conducted most of the screening experiments and corrected the manuscript; A. B. developed the protocol for the extraction of the antibiotics from the feedingstuffs; and L. H. was involved in project planning and gave further input into the presentation of the results and also corrected the manuscript.