Mode of action
Tetracyclines are part natural - obtained from types of Streptomyces - part semi-synthetic antibiotics with an extraordinarily broad spectrum of action. By blocking ribosomal protein biosynthesis, they have a bacteriostatic (growth inhibiting) action on numerous Gram-positive and Gram-negative bacteria as well as on problem germs that have no cell wall, such as Mycoplasma, Chlamydia and Rickettsia bacteria. They were the first major group of antibiotics for which the terms “broad-spectrum” was used.
Bacterial resistance mechanisms
Bacteria strains that have developed a resistance to a tetracycline are also resistant to all other members of the tetracycline family (complete cross-reactivity between all tetracyclines). As they have their effect on the ribosomes by inhibiting protein biosynthesis there, a bacterial resistance mechanism means the production of a protein that protects the ribosomes. The second important means of defence is the synthesis of a cytoplasmic membrane protein that pumps tetracycline out of the bacteria cell. The rarest resistance mechanism is chemical modification (acetylation) and thus the inactivation of tetracyclines.
Statutory residue regulation
The substances chlortetracycline, oxytetracycline, tetracycline and doxycycline are approved for the treatment of animals intended for foodstuff production. Their use requires that waiting periods be observed, which are calculated such that once the waiting period has elapsed the concentrations in the foodstuff are below the maximum residue limits. These are set out in the Appendix to Regulation (EU) 37/2010 (Table 1) and, depending on the matrix, range from 100 µg/kg (muscle, milk) to 600 µg/kg (kidney). The use of doxycycline in animals used for production of milk or eggs for human consumption is explicitly prohibited. Consequently, no residues are permitted in these foodstuffs. This so-called “zero-tolerance” also applies to honey, as tetracyclines, like all other antibiotics, are not permitted for the treatment of honey bees.
Monitoring foodstuffs of animal origin for tetracycline residues
Due to the widespread use of tetracyclines, monitoring carried out as part of the National Residue Control Plan (NRKP) encompasses virtually all animal species involved in the production of foodstuffs as well as the primary products milk, eggs and honey. To facilitate preventative consumer protection, samples are taken from the production chain, so that products that are contaminated with residues can be traced back to the farm from which they originated.
Official foodstuff-monitoring activity and cases of suspected contamination both involve testing samples from trade outlets, with monitoring also including products from non-EU countries.
Tested sample types
- Animal tissue (meat, liver, kidney)
- Aquaculture (fish, shrimps, prawns)
Range of examinations and tests
- 4-epimers of chlortetracycline, oxytetracycline and tetracycline
(Epimers are produced to a varying extent as artefacts in analytical processing and can presumably be formed in vivo as tetracycline “metabolites” in eggs at least. Along with their corresponding parent substances, they are marker residues as per Regulation (EU) 37/2010.)
Testing strategy and examination methods
For purposes of quick and cost-effective testing, samples are initially tested for possible contraventions (exceeding maximum limits or non-approved usage) using suitable screening methods. Any non-negative screening is followed up by further testing that uses a quantitative confirmation method.
- Screening methods
In animal tissue the microbial inhibitor test (5 plate test) provides an initial indication of possible tetracycline residues. The test is routinely performed on all tissue samples taken in the NRKP at the relevant AGES Institutes for Veterinary Examinations designated for testing in Graz, Innsbruck, Linz and Mödling and at the State Office for Veterinary Examinations in Klagenfurt.
Since the 5 plate test detects not only tetracyclines but also antibiotics belonging to the most varied of groups, in order to identify the antibacterial activity in cases where a positive inhibitor test is returned, post-screening is carried out using the group-specific Charm II test.
Milk, eggs and honey are analysed with the ELISA test.
- Confirmation methods
To identify and quantify substances in case of non-negative screening results, samples are analysed using high performance liquid chromatography with diode array detection (HPLC/DAD).
Prior to the chromatographic separation, samples are purified (clean-up) in a two-stage process using solid-phase extraction. This involves selective concentration of tetracyclines using metal chelate affinity chromatography and post-concentration on C18 columns.
The HPLC/DAD method is applicable for all matrices, with minor differences in the sample processing.
Detection levels are between 25 and 50 µg/kg depending on the specific substance and thus lie well below the legally stipulated maximum limits.
The Department of Veterinary Drugs, Hormones and Contaminants at the Institute for Food Safety in Vienna tests for tetracyclines in various animal matrices using the Charm II test, ELISA and HPLC and it is the National reference laboratory for tetracycline antibiotics residues.
Commission Regulation (EU) No. 37/2010 of 22 December 2009 on pharmacologically active substances and their classification regarding maximum residue limits in foodstuffs of animal origin (OJ. EU No. L 15 of 20.1.2010).