Evaluation of bacterial indicators for antibiotic test assay using resazurin-based reactions in raw milk
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Abstract
Dairy cows often develop several health issues; including bovine mastitis, diarrhea, and pulmonary diseases. Nowadays, antibiotic use is still a common practice among dairy farmers, for both therapeutic and prophylactic purposes, leading to the occurrence of antibiotic residual being left in raw milk. Hence, the detection of antibiotics in raw milk is therefore necessary. Currently, the detection kits use usually apply an assay based on bacterial growth inhibition, and most of these are a single-test assay requiring additional instruments for testing in the laboratory. This study aimed to develop a simple detection method for antibiotics in raw milk using a resazurin-based assay. Six bacterial indicators; including, Escherichia coli, Pseudomonas aeruginosa, Shigella spp., Salmonella Enteritidis, Staphylococcus aureus, and Bacillus spp. were preliminary screened for their antibiotic-resistant properties against ampicillin and ceftriaxone via minimum inhibitory concentration test. Due to the broad susceptibility to both antibiotics, only S. aureus was selected as a bacterial indicator for further study. The limit of detection (LOD) of antibiotic residues was investigated through color changes in the resazurin reaction, and a measurement of optical density at the wavelengths of 600 and 570 nm. The LOD of ampicillin and ceftriaxone residues were 1.0 and 2.0 μg/mL, respectively. Overall, a developed resazurin-based assay could be used to determine residues between 1 to 256 μg/mL for ampicillin and 2 to 256 μg/mL for ceftriaxone, which also allows for the detection of antibiotics in raw milk at room temperature; rather than at higher temperatures than the other test kits currently available.
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