Microbial growth inhibition tests are widely used as the primary screening approach for the detection of antibiotic residues in slaughter animals. In this study we evaluated and compared the performance of the European Union Four-Plate Test (EU4pt), the Nouws Antibiotic Test (NAT), and a commercial ampoule test, the PremiTest (applied to both muscle and kidney), by parallel analysis of 735 slaughter animals. The EU4pt only showed significant inhibition with two muscle samples containing 305 µg kg(-1) doxycycline and 648 µg kg(-1) tulathromycin, while an maximum residue limit (MRL) violation of 1100 µg kg(-1) sulfamethazine remained unnoticed. PremiTest-muscle only detected the sulfamethazine containing sample, all other (1.1%) suspect samples appeared false-positive results. The same test applied to kidney yielded 4.1% suspect samples, while the NAT screening (based on analysis of renal pelvis fluid) showed 4.9% suspect results. The vast majority of these samples contained tetracycline and/or aminoglycoside residues. PremiTest-kidney appeared to be more sensitive to aminoglycosides than the NAT screening, which failed to detect an MRL violation of 870 µg kg(-1) gentamicin in kidney. Detection of less than MRL levels of tetracycline residues by the NAT proved its suitability for this residue group. Whether PremiTest is sufficiently sensitive for accurate tetracycline detection in kidney remains doubtful, although changing over to kidney definitely improved the suitability of PremiTest for the detection of residues in slaughter animals.

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A non-targeted workflow is reported for the isolation and identification of antimicrobial active compounds using bioassay-directed screening and LC coupled to high-resolution MS. Suspect samples are extracted using a generic protocol and fractionated using two different LC conditions (A and B). The behaviour of the bioactive compound under these different conditions yields information about the physicochemical properties of the compound and introduces variations in co-eluting compounds in the fractions, which is essential for peak picking and identification. The fractions containing the active compound(s) obtained with conditions A and B are selected using a microbiological effect-based bioassay. The selected bioactive fractions from A and B are analysed using LC combined with high-resolution MS. Selection of relevant signals is automatically carried out by selecting all signals present in both bioactive fractions A and B, yielding tremendous data reduction. The method was assessed using two spiked feed samples and subsequently applied to two feed samples containing an unidentified compound showing microbial growth inhibition. In all cases, the identity of the compound causing microbiological inhibition was successfully confirmed. ARTICLE HISTORY

A microbiological screening method (three-plate) for the detection of the antimicrobial growth promoters tylosin, spiramycin, virginiamycin, zinc bacitracin, and avoparcin in animal feed has been developed and validated successfully. A collaborative study involving 18 laboratories receiving 172 samples was carried out to verify the performance characteristics. The detection level for tylosin/virginiamycin/spiramycin, expressed in microbiological activity, was 1 mg kg(-1) (false-positives, 2%; false-negatives, 3, 0, and 6%, respectively). Avoparcin could be detected at 1 mg kg(-1) in feed in general (false-positives, 2%; false-negatives, 0%). However, in calf feed the sensitivity was lower. The percentages of false-negatives were found to be 12%, 7%, and 0% at 1, 3, and 5 mg kg(-1), respectively (false-positives, 4%). The limit of detection for zinc bacitracin was 3-5 mg kg(-1) (false-positives, 5-10%; false-negatives, 77% at 1 mg kg(-1), 45% at 2 mg kg(-1), 12% at 3 mg kg(-1), and 4% at 5 mg kg(-1)). The method allowed for a distinction to be made between the groups of antibiotics: avoparcin/zinc bacitracin versus tylosin/virginiamycin/spiramycin. This definitely gives added value to the method in the framework of a follow-up of positive screening results by post-screening and confirmatory analysis.

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