Comparison of various assays used for detection of beta-lactam antibiotics in poultry meat

Abstract

In this study, microbiological tests for the detection of beta-lactam antibiotics in meat and meat products were evaluated. The traditional FPT (four plate test, containing Bacillus subtilis and Kocuria rhizophila), BsDA (Bacillus stearothermophilus disc assay) and a newly developed microbiological test, Premi Test (containing Bacillus stearothermophilus) were included in the study. The limit of detection (LOD) of the Premi Test was compared with the LOD of the traditional methods. The detection limits of the tests were determined by using beta-lactam antibiotic standards dissolved in meat juice, as well as meat tissue obtained from laying hens after experimental administration of amoxicillin. Positive samples, based on inhibition of growth of the organism in the test, were confirmed by high performance liquid chromatography (HPLC). Growth inhibition in the traditional tests is visible as a clear zone on the plate, whereas for Premi Test, this is based on the absence of a colour change of the test. The LODs of antibiotics tested were as follows: Penicillin G (PENG) 5 microg kg(-1), amoxicillin (AMOX) 10 microg kg(-1), ampicillin (AMP) 25 microg kg(-1), oxacillin (OXA) 30 microg kg(-1), and cloxacillin (CLOX) 30 microg kg(-1) on the plate with Bacillus stearothermophilus. Beta-lactam antibiotics can be detected also on one plate seeded with Kocuria rhizophila, although the LODs are higher: PENG 10 microg kg(-1), AMOX 25 microg kg(-1), AMP 30 microg kg(-1), OXA 50 microg kg(-1), and CLOX 50 microg kg(-1). Premi Test was performed according to the Standard Operating Procedure intended for detection of beta-lactam antibiotics in poultry tissues with following LODs: PENG 4 microg kg(-1), AMOX 5 microg kg(-1), AMP 5 microg kg(-1), OXA 40 microg kg(-1), CLOX 50 microg kg(-1). All tests are able to detect beta-lactam antibiotics such as penicillin G, ampicillin, amoxicillin, oxacillin and cloxacillin below the maximum residue level (MRL). However, the detection limits of the Premi Test for PENG, AMOX and AMP were below the limits of BsDA and the plate containing Kocuria rhizophila.

AFNOR validation of Premi®Test, a microbiological-based screening tube-test for the detection of antimicrobial residues in animal muscle tissue

“…Therefore, this study has demonstrated the capability of Premi Õ Test to detect samples at the MRL level for sulphonamides and beta-lactams, but not for tetracyclines. These conclusions have confirmed the results of different teams over recent years (Reybroeck 2000a,b;Okerman et al 2004;Popelka et al 2005). Table 7 presented the combined results of a previous study carried out in 2003 and 2004 (64 samples actually containing antimicrobial residues) with the pilot study.…”

“…Premi Õ Test has generally been compared to a reference method, focussing on a family of antimicrobials, such as beta-lactams, using spiked meat juice samples and incurred poultry muscle samples (Popelka et al 2005) or tetracyclines (Okerman et al 2004). The detection limits for various antimicrobials, calculated in spiked juice samples, were similar in the different studies.…”

Microbial screening methods for detection of antibiotic residues in slaughter animals

“…Popelka et al [46] showed that Premi®Test exhibits excellent sensitivity for penicillin, amoxicillin, ampicillin, oxacillin, and cloxacillin. The study includes results of poultry muscle samples originating from animals treated with amoxicillin.…”

Development and validation of an HPLC method for the determination of penicillin antibiotics residues in bovine muscle according to the European Union Decision 2002/657/EC

“…A significant number of methods use precolumn derivatization techniques for fluorescence detection performed by reaction with TCA (20% w/v) aqueous solution and HCHO (7% w/v) [16,19,20] or for UV detection at 320 -345 nm by benzoic anhydride to acrylate aminopenicillins and 1,2,4-triazole/imidazole and mercury(II) chloride at alkaline media (pH l 9) at 60 -658C in order to form mercury mercaptide derivatives and thus achieve higher sensitivity [21 -24]. Mass spectroscopy and MS/MS are used for confirmation analysis [25 -28].…”

“…Deproteinization of the samples can be achieved by 5% Na 2 WO 4 and 0.33 N H 2 SO 4 [12,21,22], by 2% NaCl, 5% Na 2 WO 4 , and 0.17 M H 2 SO 4 [18], by Et 4 NCl [15,27], by ACN [16], by phosphate buffer (pH 8.5) and H 2 SO 4 [24] or 30% TCA [20]. Extraction of penicillins can be achieved by CH 2 Cl 2 , CH 3 CN, and hexane [29], H 2 O, CH 3 CN, and NaCl solution [26], 2% NaCl [17,26], HCOOH [30], ACN/H 2 O (30:70 v/v) [17,18,25,26], or by tert-butyl alcohol and water [15,16].…”