Validation of a microbiological method: the STAR protocol, a five-plate test, for the screening of antibiotic residues in milk

Abstract

The results of an in-house laboratory validation of a microbiological method for the screening of antibiotic residues in milk are presented. The sensitivity of this five-plate test, called Screening Test for Antibiotic Residues (STAR), was established by the analysis of milk samples spiked with 66 antibiotics at eight different concentrations. Ten different groups of antibiotics were studied: macrolides, aminoglycosides, cephalosporins, penicillins, quinolones, tetracyclines, sulphonamides, lincosamides, phenicolated and miscellaneous drugs. It was shown that 21 antibiotics were detected by the STAR protocol at or below the maximum residue limit (MRL), and that a further 27 drugs could be detected at levels from the MRL up to four times the MRL. The sensitivity of the STAR protocol was at or below the MRL for three macrolides, one tetracycline, two aminoglycosides, some sulphonamides, half of the beta-lactams, quinolones, lincosamides, trimethoprim and baquiloprim. Moreover, the STAR protocol was at least twice as sensitive as conventional methods for macrolides, quinolones and tetracyclines. The other antibiotics had limits of detection between four and 150 times the MRL. Each plate was preferentially sensitive for one or two families of antibacterials: the plate Bacillus cereus for tetracyclines, the plate Escherichia coli for quinolones, the plate Basillus subtilis for aminoglycosides, the plate Kocuria varians for macrolides, and the plate Bacillus stearothermophilus for sulphonamides and beta-lactams. This method has been used routinely on a day-to-day basis to direct the physicochemical confirmation towards one or two families of antibiotics. Considering the high cost of liquid chromatography coupled with tandem mass spectrometry detection analyses, the reduction of the range of antibiotics to test for confirmation is a significant gain in time and money.

A plate diffusion method for detecting fluoroquinolone residues in raw cow's milk

“…The plate with the E. coli strain ATCC 11303 was sensitive, first and foremost, to fluoroquinolones, though the growth of the strain was also inhibited by other antimicrobial agents such as spectinomycin, certain cephalosporins, chloramphenicol, and colistin. Even our modified method is *The method did not display sensitivity even at a concentration twelve times the MRL > 600 µg/l sensitive enough, the results of Gaudin et al (2004) indicated that the sensitivity of the plate method with the E. coli strain according to the STAR protocol is better. A number of other studies have also focused on the sensitivity of plate diffusion methods to various antimicrobial agents during the testing of raw milk.…”

“…No inhibition of the E. coli strain around the disc was recorded even at such high concentrations. Gaudin et al (2004) published results of the validation of a 5-plate STAR method developed for screening residues of antimicrobial agents in milk. This method displayed very good sensitivity to a wide range of antibiotics (n = 66).…”

“…A new plate diffusion method with a strain of E. coli displaying sensitivity to fluoroquinolones has been developed in line with the STAR method (Gaudin et al 2004), the official method recommended by the reference laboratory of the European Community (Star Protocol 2002). The aim of this study is to determine the detection capability (CCβ) of the modified method with E. coli ATCC 11303 for fluoroquinolones registered in the Czech Republic for the treatment of diseases in cattle: danofloxacin (DANO), marbofloxacin (MARBO), ciprofloxacin (CIPRO), enrofloxacin (ENRO), and flumequine (FLU).…”

Microbiological method using Bacillus megaterium with fusidic acid for detection of macrolides in milk

“…Similarly, Tsai and Kondo (2001) detected low levels of erythromycin (50 µg/l) and achieved high minimum detectable concentrations for tylosin (390 µg/l) residues in milk when using Kocuria rhizophila. In the same way, Gaudin et al (2004) detected residues of erythromycin (30 µg/l) and tilmicosin (50 µg/l) but they did not obtain any adequate minimum inhibitory concentrations for tylosin (200 µg/l) in milk. In sheep's milk, Althaus et al (2009) determined good minimum inhibitory concentration of erythromycin (80 µg/l) and high concentration of tylosin (1000 µg/l) in Petri dish that uses the same bacteria test.…”

Comparison of detection sensitivity of five microbial inhibition tests for the screening of aminoglycoside residues in fortified milk

“…The STAR was validated for antibiotics in milk by Gaudin et al (2004). They found out that Bacillus subtilis BGA showed sensitivity to AMGs regarding the MRL, but not to all substances from the AMG group.…”