Microbiological bioassay using Bacillus pumilus to detect tetracycline in milk

Area of Science:

• Veterinary Pharmacology
• Microbiology
• Food Safety

Background:

• Tetracyclines (TCs) are vital veterinary antibiotics, but their residues in milk pose a food safety concern.
• Effective screening methods are crucial for monitoring TC residues to ensure milk safety and compliance with Maximum Residue Limits (MRLs).

Purpose of the Study:

• To develop a cost-effective, user-friendly microbiological inhibition bioassay for detecting tetracycline residues in milk.
• To optimize the bioassay for sensitivity at MRLs and assess its specificity and cross-reactivity.

Main Methods:

• A microtiter plate-based bioassay utilizing Bacillus pumilus spores and redox indicators (brilliant black and toluidine blue) was designed.
• Optimization involved determining optimal spore concentration and fortification with chloramphenicol (CAP).
• Assay performance was evaluated for specificity, detection limit (DL), and cross-specificity against other antimicrobials.

Main Results:

• The optimized bioassay used 1.6 × 10(9) spores/ml of B. pumilus and 2500 μg/L CAP in Mueller Hinton medium.
• It detected chlortetracycline, oxytetracycline, and tetracycline at 117, 142, and 105 μg/L, respectively, within 5 hours via color change.
• The assay demonstrated high specificity (97.9%), with a slight decrease in high somatic cell count milk (93.3%). Cross-specificity was low for most antimicrobials.

Conclusions:

• A sensitive and specific bioassay for detecting tetracycline residues in milk at MRLs was successfully developed.
• This method offers a practical solution for routine screening of milk for tetracycline contamination.
• The bioassay's low cross-reactivity ensures reliable detection of tetracyclines without significant interference from other common antimicrobials.