Evaluation of the performance of Bacillus cereus and Bacillus subtilis as test organisms for assay of tetracyclines and beta-lactams in chicken meat

Abstract

The uncontrolled and unrestricted use of antimicrobials may lead to the accumulation of undesirable drug residues in the treated animals and their products. To avoid adverse effects from antibiotic residue consumption such as drug resistance, regulations such as maximum residue limits (MRLs), acceptable daily intakes (ADIs) for humans and withholding times for pharmacologically active substances have been set. However, in the Kenyan poultry industry there lacks affordable, easy to perform antibiotic residues screening methods. The aim of this study was to evaluate the performance of Bacillus cereus and Bacillus subtilis as test organisms for assay of tetracyclines and beta-lactam antibiotics in chicken meat. Microbiological detection was achieved by agar well diffusion using 24 combinations of the two test bacteria, three medium pH, two poultry organs and two antibiotics. The test bacteria grew optimally at a pH range of 6-7.3. Inhibition zones increased significantly (p<0.001) when both test organisms were used to detect oxytetracycline (OTC) at a decreasing pH in both liver and kidney tissues. Zone diameters decreased significantly (p<0.001) when B. subtilis was used to detect penicillin G (PEN G) at decreasing pH from 7.3, 7.0 to 6. The zones increased significantly (p<0.001) when B. cereus was used to detect the same antibiotic in both kidney and liver samples at decreasing pH. The inhibition zone means were significantly (p<0.001) different when the effect of the organism was tested. Moreover B. cereus was more sensitive to OTC while B. subtilis for PEN G. There was significant (p<0.001) difference in the detection of PEN G in both kidney and liver samples at the different pH values although high antibiotic concentrations produced zones that were not significantly different. The inhibition zones differed significantly (p<0.001) at all OTC concentrations tested in kidney and liver. It was concluded that the pH of the growth media influences the growth of test organisms. Although OTC and PEN G were reliably detected below the MRLs, of 50ng/g in both liver and kidney for PEN G and 600ng/g in liver and 1200ng/g in kidney for OTC, by B. cereus and B. subtilis the pH of the growth media, type of organ and the test organism affected both the detection and the LODs of these antibiotics. Bacillus subtilis plate at pH 7.3 and a B. cereus plate at pH 7 can be used effectively for routine screening for residues of PEN G and OTC respectively in chicken kidney and liver tissues. The B. subtilis plate had LOD of 0.1ng/ml in both liver and kidney tissues. The LODs were 131.3ng/ml and 33.4ng/ml, in liver and kidney respectively, on the B. cereus plate. This screening test is technically simple and can be carried out in any laboratory.