M.G. Pikkemaat

Microbial screening methods for detection of antibiotic residues in slaughter animals

AbstractMonitoring of food products from animal origin for the presence of antimicrobial residues is preferably done using microbial screening methods because of their high cost-effectiveness. Traditionally applied methods fail to detect the maximum residue limits which were established when EU Council Regulation 2377/90 came into effect. Consequently, during the last decade this has led to the development of improved microbial screening methods. This review provides an overview of the efforts expended to bring antibiotic screening methods into compliance with EU legislation. It can be concluded that the current situation is still far from satisfactory. FigureMicrobial inhibition assay for muscle samples

Comparison of three microbial screening methods for antibiotics using routine monitoring samples

Monitoring large numbers of slaughter animals for the presence of antimicrobial residues is preferably carried out using microbiological screening methods, because of their high cost-effectiveness. An evaluation of the Nouws antibiotic test (NAT) was performed on routine monitoring samples and the performance of the method was compared with two other microbial screening methods: Screening test for antibiotic residues (STAR) and Premi Test. Analysis of 591 samples yielded four MRL violations. Three of them concerned tetracyclines that were only detected with the NAT and the STAR method. The fourth, 172 microgkg(-1) Sulfadiazine, was detected by all three methods. Additionally, 156 microgkg(-1) Tulathromycin was found in porcine meat, while for this residue no MRL in muscle has been established.

Improved microbial screening assay for the detection of quinolone residues in poultry and eggs

An improved microbiological screening assay is reported for the detection of quinolone residues in poultry muscle and eggs. The method was validated using fortified tissue samples and is the first microbial assay to effectively detect enrofloxacin, difloxacin, danofloxacin, as well as flumequine and oxolinic acid, at or below their EU maximum residue limits (MRL). The accuracy of the assay was shown by analysing incurred tissue samples containing residue levels around the MRL. Liquid chromatography–tandem mass spectrometry (LC–MS/MS) quantification of the quinolone concentration in these samples showed that the test plate can be used semi-quantitatively, allowing the definition of an “action level” as an inhibition zone above which a sample can be considered “suspect”. The presented assay is a useful improvement or addition to existing screening systems.

Application of a luminescent bacterial biosensor for the detection of tetracyclines in routine analysis of poultry muscle samples

Tetracyclines are extensively used in veterinary medicine. For the detection of tetracycline residues in animal products, a broad array of methods is available. Luminescent bacterial biosensors represent an attractive inexpensive, simple and fast method for screening large numbers of samples. A previously developed cell-biosensor method was subjected to an evaluation study using over 300 routine poultry samples and the results were compared with a microbial inhibition test. The cell-biosensor assay yielded many more suspect samples, 10.2% versus 2% with the inhibition test, which all could be confirmed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Only one sample contained a concentration above the maximum residue limit (MRL) of 100 µg kg−1, while residue levels in most of the suspect samples were very low (<10 µg kg−1). The method appeared to be specific and robust. Using an experimental set-up comprising the analysis of a series of three sample dilutions allowed an appropriate cut-off for confirmatory analysis, limiting the number of samples and requiring further analysis to a minimum.

Screening methods for the detection of antibiotic residues in slaughter animals: comparison of the European Union Four-Plate Test, the Nouws Antibiotic Test and the Premi®Test (applied to muscle and kidney)

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 Premi®Test (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. Premi®Test-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. Premi®Test-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 Premi®Test is sufficiently sensitive for accurate tetracycline detection in kidney remains doubtful, although changing over to kidney definitely improved the suitability of Premi®Test for the detection of residues in slaughter animals.

Are antibiotic screening approaches sufficiently adequate? A proficiency test

A proficiency test including the screening analysis of antibiotics in beef using cryogenicly minced materials was organized by RIKILT in 2009. The test included blank beef samples and beef samples spiked with either flumequine or a combination of lincomycin and spectinomycin around the maximum residue limits [1]. The suitability of the materials was demonstrated with a homogeneity and a stability study. This study showed that cryogenically minced spiked muscle material is suited for proficiency tests aiming at the screening and the confirmatory analysis. Of the 26 participants, 23 carried out their in-house screening approach involving microbial, biochemical or instrumental methods, or a combination of these to cover the broad range of antibiotic groups. The false negative rate was 73% for microbial methods, 50% for biochemical and 22% for instrumental methods. These results indicate that substantial effort is needed to improve screening approaches and that more regular proficiency tests are needed to reveal the shortcomings in the currently applied screening methods.

Non-targeted workflow for identification of antimicrobial compounds in animal feed using bioassay-directed screening in combination with liquid chromatography-high resolution mass spectrometry

ABSTRACT 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. Graphical Abstract

Revision of the National Residue Control Plan - application on the red meat supply chain

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