Detection of antibiotic residues in animal-derived foods using Bacillus subtilis based STAF test

Rafique, M.A. and Khalid, K. and Farooq, Z. and Bashir, I. (2026) Detection of antibiotic residues in animal-derived foods using Bacillus subtilis based STAF test. ACTA ALIMENTARIA: AN INTERNATIONAL JOURNAL OF FOOD SCIENCE, 55 (3). pp. 466-475. ISSN 0139-3006

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Abstract

Widespread antibiotic use in livestock raises concerns about residues in food, which can contribute to toxicity and antimicrobial resistance. This study applied the Swab Test on Animal Food (STAF) with Bacillus subtilis . A total of 80 samples (20 each of raw milk, beef, mutton, and chicken meat) were collected aseptically from slaughterhouses and retail markets in the east-central region of Pakistan. The STAF protocol involved nutrient agar supplemented with dextrose, seeded with B. subtilis spores; sterile swabs were immersed in milk or inserted into meat samples, then placed on prepared plates alongside a neomycin control disc, and incubated at 37 °C for 16–18 h. Inhibition zones ≥2 mm around the swabs were considered positive for antibiotic residues. Out of 80 samples, 21.25% were positive: 14 out of 20 milk samples (70%), 1 out of 20 beef (5%), and 2 out of 20 chicken (10%); with no residue detected in mutton (0/20). On nutrient agar with 0.4% dextrose, STAF reliably detected inhibition zones of ≥2 mm. However, it is important to emphasise that the STAF test is a qualitative screening method; positive results indicate the possible presence of antimicrobial residues rather than definitive evidence of regulatory non-compliance. Therefore, the STAF method is a practical, low-cost screening tool for detecting antibiotic residues in resource-limited laboratories, however, confirmatory quantitative analysis using chromatographic methods is essential to detect antibiotic concentration, class, and limits above or below the maximum residue limits (MRLs).

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