introduction

Tetracyclines are the most commonly used antibiotic class in animals used for food production. They are also used in agriculture and medicine. In the USA tetracyclines for animals comprise 43% of the 13 million kg imported and 4% of 3.3 million kg of the human use. New Zealand uses about 4,616 kg tetracycline, mainly oxytetracycline/year.

The graph below shows the use of the different antibiotic classes in animals in Europe and in Australia. It shows that tetracyclines are the second most common class of antibiotics sold with the beta-lactams being the most common. With its policies, Europe has been able to reduce to antibiotics sold for food animal use by 50%. Of the tetracycline sales, Spain, Poland and Italy rank the highest and Iceland and Norway ranking the lowest.

Tetracyclines are 1st tier antibiotics and commonly used in food animals as they are of low importance for human treatments. See Importance Ratings and Summary of Antibacterial Uses in Human and Animal Health in Australia, Version 1.0 (2018). Online ISBN: 978-1-76007-369-5. However, it must be noted that in hospitals implementing good antibiotic practices, tetracyclines were the only antibiotic where the prescriptions increased. They are now the 5th most prescribed class of antibiotics. The first 4 are beta-lactam antibiotics.

To find out more about the tetracyclines consult: Merck Veterinary Manual or Chopra I &Roberts M (2001). Tetracycline Antibiotics: Mode of Action, Applications, Molecular Biology, and Epidemiology of Bacterial Resistance. Microbiology and Molecular Biology Reviews, 65:232-260

The different types of tetracyclines and their use

The tetracyclines in bold are those used routinely in veterinary medicine

All tetracyclines have a typical 4-ring structure. They differ in the side groups of molecules.

Classes of tetracyclines

1. Natural as extracted from different Streptomyces species (a branching, filamentous, gram-positive, soil bacterium). They are also short-acting (T_½ = 6-8 hours).

• Oxytetracycline
• Chlortetracycline (Aureomycin was first tetracycline that was commercially available in 1948).
• Tetracycline

2. Semi-synthetic and Long-acting (T_½ > 16 hours). Doxycycline is registered for animal and human use. Minocycline is used in people. These are more lipophilic.

3. Newer semisynthethetic and synthetic cyclines i.e. the semisynthetic  tigecycline and omadacycline and synthethetic eravacyline were developed to overcome antibiotic resistance to the tetracyclines. Only used in people. They are considered of high importance to people and can only be used off-label in individual animals for resistant infections.

SPECTRUM OF ACTIVITY AND Uses of tetracyclines

Tetracyclines are active against both gram-positive and gram-negative bacteria and certain protozoa – those that lack mitochondria. Not being dependent on oxygen for transport into bacteria, they are also active against obligate anaerobes. They are very broad-spectrum and can be used to treat most infections.

They are specifically used for respiratory tract infections in livestock, internal abscesses, mycoplasma infections and foulbrood in bees. In people, they are used to treat acne, brucellosis, tuberculosis and Helicobacter infections.

2. They are also able to enter animal cells to attack intracellular bacteria and protozoa.

They are used to treat intracellular infections in animals and humans targeting Rickettsial, Anaplasma, Coxiella burnetti (Q-fever), Chlamydia, Brucella, Erhlichia and Plasmodium infections.

3. Their ease of oral administration has allowed them to be used for mass medication or prophylactic treatment in livestock and trees and seeds. In the past, they were used as antibiotic growth promoters. They are in most countries banned as growth promoters.

4. They are 1st tier antibiotics in animals. This is because they are only used to treat chronic and non-life-threatening diseases in people. They are the favoured antibiotic in aquaculture.

5. Since they are bacteriostatic, they should be used more for chronic disease in immune competent animals.

6. They are cheap, thus affordable for use in food animals.

​​​​​​​7. They are comparatively safe and can be used in all animals. (See adverse effects)

8. They are also matrix metalloprotease inhibitors and have been used as anti-inflammatories, anticancer therapy or bone and breast tumours, immune modulators in immune-mediated dermapathies in dogs and plasmacytic pododermatitis in cats. It can be used to treat recurrent corneal ulceration. They are useful in the latter as they inhibit collagenases. They are also used to lengthen digital flexor tendons in foals with congenital contracture of these tendons.

9. They reduce the adverse events and improve killing of microfilaria when administered with heartworm-cides.

9. Being strong chelators, they bind to bone undergoing mineralisation. Tetracyclines also fluoresce under UV light. So researchers can easily detect bone growth, whether bait containing vaccines has been ingested by wildlife etc.

The Table below shows the use of tetracyclines in animals

To find out which antibiotics are registered in Australia for animal use go to the PUBCRIS website.

• Tetracycline, Oxytetracycline and Chlortetracycline are registered for oral use in pigs, poultry, other avians and fish, parental use in cattle, sheep, goats and horses
• Topical – eye
• Doxycycline for dogs, cats and caged birds

Withdrawal Periods in Australia

Before administering tetracyclines to food animals, make sure you know what the drug withdrawal times are. I don’t expect you to learn them, but you should apply this knowledge when treating food animals with antibiotics. Below is a summary:

Varies: read package inset

• Extended to 30 days for export animals
• 90 days for animal products destined for Russia

Oral Preparations:

• Prophylactic dose: Broilers : 2 days before slaughter; Pigs and calves: 5 days before slaughter
• Therapeutic dose oral: Calves: 10 days; Broilers & Pigs: 7 days; Horses: 28 days; Milk: 3 days, Poultry: meat 4 days, Only chlortetracycline registered for layers eggs: 0 days
• ​​​​​​​Therapeutic dose parenteral: Cattle: 14 days before slaughter, Sheep & Pigs 10 days before slaughter; Milk: 3 days (short-acting).

Laboratory Detection of Tetracyclines (For information ONlY)

The presence of active tetracyclines in animal products is strictly controlled and thus maximum residue levels are measured by authorities.

• • Microbial: Not specific to tetracyclines
  • Immunoassays: Can’t detect all tetracyclines
  • Switching on of luminescent reporter genes in E. coli containing recombinant plasmids with the tetracycline operon
  • HPLC with UV-light to detect fluorescence

pharmacodynamics of the tetracyclines

Absorption,  Distribution and Excretion of the Tetracyclines

Most tetracyclines generally have excellent intestinal absorption, except ruminants.

Thus they can be administered orally, systemically and applied topically. However, only oxytetracycline is non-irritant to tissue and is the only one that can be administered intramuscularly or subcutaneously. The others will cause sterile granulomas (abscesses). When administering intravenously, also administer slowly or diluted to avoid cardiovascular collapse.

Doxycycline being the most lipophylic of the tetracyclines is 100% absorbed, followed by tetracyclines, oxytetracyclines and lastly chlortetracycline at 25% absorption (see graph). It takes between 2 to 4 hours before maximum concentrations of the tetracyclines are reached in the blood after oral administration.

Tetracyclines are also strong ionic chelators, which means that diet will affect their intestinal absorption. They bind strongly to calcium, magnesium, iron and aluminium. So this antibiotic should not be administered in foods rich in these ions as they can decrease absorption by as much as 50%. Of the tetracyclines mentioned, doxycycline is the weakest ionic chelator. They are most active at pH 6 to 6.5 where they are more soluble. So don’t administer with antacids.

Once in the body, the tetracyclines become protein-bound with doxycycline being more than 90% protein-bound and oxytetracyline only 30% protein-bound. Thus doxycycline has the longest T½ and oxytetracycline the shortest. The T½  varies from 5.5 to 11 hours. These antibiotics will be short-acting and more active in animals with low albumen levels due to protein starvation or liver disease.

Tetracyclines have a moderate tissue distribution with the more lipophilic doxycycline distributing the best. They diffuse well into the sputum, urine, peritoneal and pleural fluids. Whilst they can cross the blood-placental barrier they penetrate the CSF poorly – only 10 – 25% of the blood levels. Inflammation of the blood-brain barrier will allow better penetration of the tetracyclines. The tetracyclines being ionic chelators concentrate and persist in areas of active bone deposition and in developing teeth. The older tetracyclines will concentrate in the eye. Tetracyclines also concentrate in the liver where doxycycline is concentrated 5x more than the blood. Although tetracyclines distribute well to the mammary glands, they chelate with the calcium and magnesium ions and tend not the be excreted via the milk.

Doxycycline is mainly excreted by the biliary system in the liver and the other tetracyclines are both excreted through the biliary system and the kidneys.

Tetracyclines are time-dependent antibiotics with a persistent effect. This means that their drug concentrations should be above the MIC at 24 hours after administration.

Mode of action of the tetracyclines

How do tetracyclines access bacteria, especially intracellular bacteria?

Tetracyclines being moderately lipophilic are able to diffuse across eukaryotic cell membranes across a concentration gradient. To cross the bacterial outer membrane, they chelate with magnesium that allows them to move through the energy dependent membrane channels. Once in the periplasmic space they disassociate from the magnesium and diffuse across the bacterial cell membrane. ​​​​​​​They again chelate with magnesium ions to enable them to bind to the 30S ribosome.

Mode of Action of Tetracyclines

Tetracyclines reversibly bind to the 30S ribosomal subunit where they prevent the binding of aminoacyl-tRNA to the of the mRNA-ribosome complex. Translation for the production of essential cell proteins does not occur. All tetracycline members of this antibiotic class have similar attachment sites in the 30S ribosome and the same mode of action. Thus bacteria that develop resistance to for example doxycycline tend to become resistant to all tetracycline antibiotics = cross-resistance.

Drug interactions

• Chloramphenicol (transacetylase) & tetracycline = additive
• Penicillins & tetracyclines – antagonistic? Theory yes, but the effect is not noted clinically;
• Tetracyclines & novobiocin – synergistic = improved cell uptake = increased spectrum
• Tetracycline binders in food – dairy products, bone meal high in calcium and magnesium; products rich in iron
• (in people tetracyclines can reduce the absorption of contraceptives and interact with a number of drugs include anticoagulants)

Adverse effects and contraindications of the tetracyclines

Despite being relatively safe and can be used in all species of animals, there are risks.

1. Being broad-spectrum and able to be administered orally there is a great risk that they will cause dysbiosis. This effect is especially noted in ruminants and horses. Note that in animals with an active rumen, that tetracyclines will be used up for the resident microflora and won’t be absorbed. So don’t administer tetracyclines orally in these animals. Doxycycline has an enterohepatic circulation and thus can have a deleterious effect on the intestinal microflora.

2. Oral administration and excretion in active format in the urine and faeces means that they will be in non-bacteriostatic concentrations when in contact with normal microflora. Thus bacterial resistance to this class of antibiotics is common. (See antibiotic resistance notes).

3. Environmental contamination by tetracyclines with increased resistance in environmental bacteria. (See antibiotic resistance notes). This can be common, due to the widespread use of tetracyclines in aquaculture, food animals and especially agriculture.

4. Being strong ionic chelators they bind to new mineral deposits in bone and enamel. At these points the bone and enamels is weak. Thus tetracyclines should be used with care in late pregnancy and growing animals. Human foetuses and Infants taking tetracyclines can develop a dark brown line along their teeth (see picture).

5. They are unstable in light breaking down to brown toxic epi-anhydrotetracyclines. This can lead to acquired Fanconi syndrome (kidney tubule disease) and glucosuria in people.   This does not happen with doxycycline.

6. Intravenous solutions should be diluted in sterile Normal saline or administered slowly to avoid cardiovascular collapse. Intravenous doxycycline has caused arrhythmia and sudden death in horses.

6. Distribution of tetracyclines to the skin can in thin-skinned people cause photosensitisation with severe sunburn. This effect has not been recorded in animals.

7. High doses can lead to hepatotoxicity and renal toxicity (not doxycycline).

8. Tables can cause oesophageal ulcerations and strictures in cats. Tablets can get stuck mid-cervical oesophagus. As they dissolve their acidity will irritate the mucosal lining of the oesophagus causing erosion, ulcers and strictures. This usually happens days to weeks after starting treatment.  Administer liquid forms of doxycycline. However, if only tablets are available then giving 5 mL fluid after the tablet has been administered reduces this effect. Rather administer doxycycline monohydrate which is less acidic than doxycycline hyclate.

mechanisms of antimicrobial resistance in the tetracyclines

1. Tetracycline Efflux: Under the control of plasmid (motile) genes (primarily tetA and

   Learning Objective

   Describe the mechanisms of antimicrobial resistance in the tetracyclines

    

   tetB for the gram-negatives and tet(K) and tet(L) for the gram-positive bacteria), the bacteria produce tetracycline-specific energy-dependent efflux pumps, that pumps the tetracycline out of the cell straight after it enters, prevent an accumulation of tetracycline within the bacterial cell. This confers high-level resistance.  A way to limit access of tetracycline to ribosomes is to reduce intracellular concentrations of tetracycline by pumping it out of the cell at a rate equal to or greater than its uptake. Most common resistance mechanism in this class of antibiotics. Certain strains bacteria like Klebsiella pneumoniae develop mutations in genes that stimulate a bacterial “stress response”. This stress response results in the upregulation of intrinsic multidrug efflux pumps.

2.  Ribosomal Protection: Protoplasmal proteins that changes the ribosomes conformation making them insensitive to the action of tetracycline. The genes for ribosomal protection have been found on plasmids and self-transmissible chromosomal elements on both gram-positive and gram-negative bacteria.  The best characterised are Tet(O) and Tet(M).
3.  ​​​​​​​Enzymic Inactivation: The tet(X) gene found in some strains of E. coli enzymatically alters tetracycline in the presence of oxygen and NADH.
4. Alteration of target. Mutations can lead to alterations in the tetracyclines binding sites on the 30S ribosome. preventing tetracyclines from binding. An example is: Mycoplasmopsis bovis an agent involved in the bovine respiratory disease complex.