INTRODUCTION

In this chapter you will learn about the different beta-lactam antibiotics, especially the penicillins and cephalosporins. All beta-lactams are active against the bacterial outer membrane (cell wall). Also active against the bacterial outer cell membrane are vancomycin, bacitracin, phosphomycin and cycloserine. Of the latter, the only antibiotic commonly used in veterinary medicine is bacitracin where it is incorporated into animal feed. Its use in animals has gradually been declining.

THE BETA-LACTAM ANTIBIOTICS AND OTHER WALL-ACTIVE ANTIBIOTICS

Beta-lactam antibiotics are those antibiotics that have a beta-lactam ring as part of their core structure. This group of antibiotics include penicillins, cephalosporins, carbapenems and monobactams. The diagram below indicates the different classes of beta-lactam drugs and shows their basic structure.

The beta-lactam antibiotic group contains the largest number of antibiotics that are widely used, especially the penicillins and cephalosporins. They are increasingly becoming the largest group of antibiotics used in veterinary medicine. In fact, in Europe, they are. Examining the graph below, shows that whilst the penicillins are used widely both in food and non-food animals, cephalosporins are used predominantly in companion animals. However, in recent years there has been a trend to increase the use of cephalosporins, especially ceftiofur in food animals – not shown in graph below. This trend is seen as alarming as this antibiotic is a 2nd tier.

the penicillins and their spectrum of activity

It has been known for centuries that certain moulds produce antibacterial substances and scientists in the late 19th Century used the mould to treat infections. However, interest in it only occurred in 1928 when Alexander Fleming isolated penicillin from Penicillium and used it to successfully treat a nasal sinus infection of his assistant. Florey, Chain and Fleming received a Nobel Prize for their early work on the bacteriocidal and treatments of penicillin. However, penicillin could only be used widely when researchers were able to successfully stabilise penicillin and produce it in bulk. This happened in the mid 1940s. Dorothy Hodkin’s elucidation of the chemical structure of penicillin in 1945 paved the way for the chemical manufacture of penicillin in 1957 and its synthetic derivatives.

It was during the Second World War when penicillin was able to save hundreds of lives, especially amputee victims and persons suffering from sexually transmitted diseases (STDs).

Natural penicillins have a predominantly gram-positive spectrum of activity. It was discovered early on that bacteria develop resistance to penicillin and thus the penicillnase resistant penicillins were developed. A broader spectrum of activity, intestinal absorption and increase of half-life was achieved by manufacture of the aminopenicillins. Amoxycillin is still used today as a first tier antibiotic. The extended spectrum penicillins such as ticarcillin were developed to provide a relatively safe way to treat antibiotic resistant bacteria such as Pseudomonas aeruginosa. However, these drugs could not be administered orally and have a short half-life. When it was discovered that bacteria could enzymically destroy the beta-lactam ring, the beta-lactams were combined with a minor antibiotic known as clavulanic acid. Clavulanic acid is able to protect the beta-lactam ring against the action of beta-lactamases produced by gram-positive and gram-negative bacteria.

Most penicillins have a good spectrum of activity against obligate anaerobic bacteria.

Developments and the different classes of penicillins

1. Natural : Penicillin G, Penicillin V
2. Pencillinase resistant: methicillin, oxacillin, cloxacillin
3. Aminopenicillins: amoxicillin (potentiated with clavulanic acid)
4. Extended spectrum penicillins (anti-Pseudomonas): carbenicillin, ticarcillin, pipercillin
5. Potentiated penicillins: amoxicillin plus clavulanic acid; ticarcillin plus clavulanic acid

Spectrum of activity of the penicillins

The cephems: cephalosporins and cephamycins

Cephalosporins produced by the mould Acremonium were first discovered in 1945 and sold in 1964. Together with the cephamycins they are part of a group known as the cephems.

Earlier cephalosporins such as cephalexin have a predominantly gram-positive spectrum of action and are thus often used as first tier therapy in skin infections. They are also active against some gram-negative bacteria. The good palatability and oral absorption of cephalexin has lead to it being commonly used in companion animal medicine.

They are traditionally divided into generations, which mirrors the development of the antibiotics over time. However, the generations are now associated with spectrum of activity, with 1st generation cephalosporins more gram-positive with some gram-negative spectrum, 2nd generation cephalosporins are broad-spectrum and 3rd generation cephalosporins are predominantly gram-negative and contain members that are reserved for multi-drug resistant gram-negative bacteria such as Pseudomonas aeruginosa. Fourth generation cephalosporins are extended spectrum similar to that of 1st generation cephalosporins, however they are more resistant to ESBLs and can penetrate into the brain. Cefquinome (Cobactan) is registered in the USA to treat respiratory disease in cattle. Application for use in Australia was submitted in 2015.

Cefovecin (Convenia) is in development a 3rd generation, but has the spectrum of a 1st generation cephalosporin.

Cephamycins are generally not used in veterinary medicine. However there are two of interest, cefoxitin and nitrocefin (rapid chromogenic test) which are used to detect methicillin resistant Staphylococcus aureus and methicillin-resistant coagulase negative staphylococci. Cefoxitin also unlike the other cephalosporins and cehamycins is more effective against obligate anaerobes.

Cephalosporins should not be used to treat infections caused by Enterococcus and Clostridium/ Clostridioides species.

 the carbapenems

Originally isolated from the bacterium Streptomyces. The synthetic carbapemens: meropenem and imipenem are reserved for the treatment of multi-drug resistant gram-negative infections. They are especially useful to treat infections caused by Pseudomonas aeruginosa. None of the carbapenems are registered for animal use in Australia. They can only be used extralabel for multidrug resistant gram-negative infections and should not be used in food animals.

Vancomycin (Since it is not registered for veterinary use, you will not be examined on it).

​​​​​​​This is a glycopeptide antibiotic that is similar to an antibiotic growth promoter that was used in the 20th Century known as Avoparcin. 

• Binds to the peptide branches preventing cross-linking
• It is narrow spectrum targeting gram positive bacteria, especially Staphylococcus, Streptococcus and Enterococcus. It is also effective against Clostridium species.
• It is not active against gram-negative bacteria as this large molecule cannot cross the lipopolysaccharide layer.
• It is not recommended for veterinary use. So any use in animal would be extralabel

• Its clinical uses include:

Mechanisms of action of the beta-lactam and other outer membrane active antibiotics

All beta-lactam antibiotics must enter the bacterial cell to exert their effect. Thus their first barrier is the outer cell membrane. Being hydrophilic, the beta-lactam antibiotics diffuse easily across the outer peptidoglycan layer of the gram-positive bacteria. However, they have to be transported by porins in the lipoprotein layer of the gram-negative bacterium. The ability to cross these barriers is the primary reason for the variation in antibacterial spectrum demonstrated by the various groups of beta-lactam drugs.

Irrespective of the of the beta-lactam antibiotic all exert their effect by preventing transpeptidation (cross-linking) of the peptidoglycan layer in the outer membrane of gram-positive (thick layer) and gram-negative (thin layer) bacteria.

To understand this better, a brief overview of peptidoglycan layer manufacture is provided and the sites where the different antibiotics act are also shown.

Beta-lactam antibiotics prevent the action of the enzymes (co-called penicillin-binding proteins) that catalyses cross-linking between the peptidoglycan layers.  The higher osmotic pressure within the cells places pressure on the uncross-linked peptidoglycan layers that slip over each other until they rupture causing bacterial cell lysis and death. (Link to YouTube video showing this). Thus all the beta-lactam antibiotics are bacteriocidal and useful in the trreatment of immune-compromised patients and in peracute infections when rapid bacterial killing is critical for patient survival. Since only bacteria have a peptidoglycan layer, the beta-lactam antibiotics are relatively safe to use in animals and people. There are exceptions (refer to adverse effects).

Beta-lactams require metabolising bacteria to be effective.

Absorption,  Distribution and Excretion of the Beta-Lactam Drugs

Intestinal absorption

Many of the penicillins and cephalosporins are hydrophilic and thus poorly absorbed after oral administration. Beta-lactams with moderate oral absorption are:​​​​​​​

• Amoxicillin (Amoxycillin) – it is better absorbed than the similar ampicillin and is stable in stomach acid. Oral absorption slightly improved with food
• Ampicillin – fasting oral absorption better
• Penicillin V
• Cephalexin – acid stable. Food has no effect on absorption

Oral absorption of amoxicillin is enhanced if administered with food. The graph below showing the serum concentration of amoxicillin over time after oral administration in dogs illustrates this point. Stating that the package insert will state that it does not matter. Ampicillin is better absorbed when fasting and food intake has no effect on the absorption of cephalexin.

​​​​​​​Thus these antibiotics are often administered parenterally (intramuscular/subcutaneous/intravenous) or topically (intramammary/eye).

Note on beta-lactam drug storage

After reconstitution many of the beta-lactam drugs have a short shelf-life and will reduce in potency rapidly – so don’t use them beyond their expiry date – it will lead to sub-therapeutic concentrations of antibiotics being administered.

Distribution and metabolism of the beta-lactam antibiotics

Once in the body the beta-lactam antibiotics distribute to aqueous compartments.Their duration above the minimum inhibitory concentration (MIC) is predominantly a time-dependent effect, as illustrated in the graph below of the serum concentrations of cefotaxime when administered sub-cutaneously to dogs.

Most of the penicillins and cephalosporins have a short half life in the body and thus have to be administered often (time-dependent) to maintain serum concentrations. This is especially true for the antipseudomonal drugs like ticarcillin or ceftazidime. This is illustrated in following graph after ticarcillin has been administered intramuscularilly in dogs. The half-life of ticarcillin is 2 hours. Note its time-dependency.

Since these antibiotics are extremely useful, the inconvenience of the short half-life as led to the development of a number of different formulations including depot and protein-bound formulations. Both result in the slow release of active antibiotic over time.

• Procaine (related to lignocaine, a local anaesthetic) is a depot preparation allowing slow release of penicillin in the blood. It is a banned substance in race horses as procaine is a pain killer and can cause excitation – detectable for 2 weeks. Should not be used in pocket pets, reptiles and birds. To prolong the T½ further benzathine an insoluble salt that stabilizes benzylpenicillin is added to the Procaine penicillin. Benzathine penicillin should not be administered without procaine in horses as it does not reach therapeutic concentrations in the blood.
• Another depot preparation is ceftiofur crystalline free acid (CCFA) that is administered subcutaneously in the ear of cattle, intramuscular in horses and pigs.  It differs from ceftiofur sodium by having a 4 times longer T½ and does not reach high plasma concentrations (see graph below). Its depot activity is associated with the ceftiofur being in an oil-base.
• Depot preparations should never be administered intravenously.
• ​​​​​​​The graphs below show the change in half-life procaine has made to penicillin, adjusting it from 3 to 6 hours to 12 to 26 hours.

Cefovecin (Convenia) is a long-acting cephalosporin that is registered for use in cats and dogs. It is highly protein bound (99% in cats) slowly releasing active antibiotic. In cats, its half-life is 6.9 days and dogs 5.5 days. Thus the manufacturers recommend dosing every 14 days to treat bacterial infections where the drug concentrations will remain above the MIC. It is effective against the streptococci and staphylococci and is used to treat skin infections. However, some staphylococci, Pasteurellaceae and Enterobacteriales may have higher MICs which may require a higher treatment dose or repeated treatment every 7 days. This is especially of concern in dogs with the shorter half-life of cefovecin. Thus empirical treatment of urinary tract infections in dogs where E. coli is the main culprit, should be every 7 days.

In birds and reptiles the protein binding of cefovecin is much less and thus the half-life is 1 to 3 hours. So it is not suitable as a long-acting formulation for these animals.

One of the primary concerns about the long-acting formulations is that they will continue to release antibiotic when the antibiotic concentrations are below the MIC of the infecting bacteria. Thus any surviving bacteria especially those with high MICs may be stimulated to develop resistance. Cefovecin should be a 2nd tier antibiotic that is only used when the laboratory antibiotic susceptibility results are available.

Beta-lactam metabolism and excretion

• Most formulations don’t break down in the liver and some that do like ceftiofur break down to an antibacterial desfuroylceftiofur, so the nett effect is the same.
• Protein unbound beta-lactam drugs are actively excreted by the kidneys with pharmacologically active antibiotics accumulating in the urine.
• There is some biliary excretion
• Short (gram negative) to intermediate (gram positive) post-antibiotic effect of penicillins and cephalosporins. Carbapenems have a long post-antibiotic effect.

Withdrawal Periods

Withdrawal periods in food products are regulated to ensure that people are not exposed to low concentrations of antibiotics that may stimulate an allergic response. Penicillin hypersensitisation is one of the more common allergies in people. There is also a risk that the intestinal microflora of people may develop antibiotic resistance after being exposed to low concentrations of antibiotics.

Residue test: Mass spectrophotometry and HPLC methods are more accurate. A rapid immunochromatography test is available: Charm SL6™Beta-lactam Test for Amoxicillin, Ampicillin, Ceftiofur, Cephapirin, Cloxacillin and Penicillin G residues in milk.

Some withdrawal periods are shown below (Don’t learn them). Be aware that you must know what they are before prescribing any for food animals. Essentially the longer acting the formulation the longer the withdrawal period. Ceftiofur is interesting, in that it has a 0 day withdrawal period in milk. This is because it does not cross the blood-mammary barrier. This does not mean, however, that the antibiotic won’t enter the udder when there is mastitis as inflammation may reduce the efficacy of the blood-mammary barrier.

Procaine/benzathine penicillin – long acting – procaine

• Meat: Do not use less than 5 days before slaughter for human consumption
• Milk: Milk collected from animals within 36 hours (single dose) or 72 hours (multiple dose) following treatment. This milk should not be fed to bobby calves.

Ceftiofur

• WHP: Meat: Cattle: 24 hours, Horses: 28 days.
• Milk: Cattle: 0 days (does not cross blood-udder barrier)

Intramammary  – most are beta-lactam drugs (cloxacillin, amoxicillin, amoxyclav, cefuroxime)

• Milk: IN LACTATING 72 – 96 hours
• ​​​​​​​Meat: 30 days

drug interactions

Antibiotic interactions

Synergism

Due to their unique mechanism of action, the beta-lactam antibiotics often act synergistically with other antibiotics and are often used in combination therapies.

A general rule of thumb is that if they have different targets, they are more likely to be synergistic or have no co-effect. The same target and they will be antagonistic. Avoid using two beta-lactam antibiotics in combination therapy unless you know they target different penicillin binding proteins.

• A common combination therapy is a beta-lactam drug with an aminoglycoside. The most common antibiotic used in horses is a penicillin-gentamicin combination. The combination not only widens the antibacterial spectrum, but the penicillin aids the aminoglycoside in accessing the bacterial cytosol. The aminoglycosides aid the beta-lactam drugs by binding to beta-lactamases.
• Another common combination therapy that is used a lot in small animal practice is combining a beta-lactam drug with either clavulanic acid or subactam. Both clavulanic acid and sublactam have a weak antibacterial activity, but when combined with a beta-lactam they will protect the beta-lactam antibiotic from beta-lactamases. Amoxicillin plus clavulanic acid (amoxyclav) is the most commonly used antibiotic in small animal practice.
• Because they are both bacteriocidal and target different sites in the bacteria the beta-lactams and fluoroquinolones act synergistically.

Antagonism

In theory beta-lactam antibiotics and tetracyclines should be antagonistic. Although they have different targets, tetracyclines impair cell metabolic processes that may negatively affect the action of the beta-lactams. However, in practice, the antibiotics exert little effect on each other. So even though they may be co-administered, there is no advantage in combining them as recovery from infection is the same whether the antibiotics were used alone or in combination.

When administering drugs that are excreted through the renal tubules i.e. some antiinflammatories, there may be competition for excretion with beta-lactams. This will increase the half-life of the beta-lactams or the other treatments.

Adverse and Toxic Effects of the beta-lactam antibiotics

In animals there are fewer recordings of adverse effects after the administration of beta-lactam antibiotics compared to other antibiotic classes.

1. Common with other antibiotics, is the negative effect on the gastrointestinal microflora where dysbiosis can lead to diarrhoea. In people, the administration of amoxicillin is associated with Clostridium difficile diarrhoea. Diarrhoea is more common in the hind gut fermenters like horses and lagomorphs (rabbits and hares). Link to article that reviews Clostridium difficile colitis in horses.

2. The most serious effect are hypersensitivity reactions which vary from anaphylaxis, bleeding disorders and skin rashes. Although, this is more common in people. However, it occurs in dogs and horses. Any animals showing a mild adverse response to the beta-lactam antibiotic including skin swelling, shivering & vomiting should be provided with an alternative antibiotic. Whilst most animals will only be hypersensitive to the penicillins, some may show a broader beta-lactam allergy.

3. False positive urine glucose – some tests

4. Whilst the beta-lactam antibiotics have a wide therapeutic index, overdose can cause renal failure.

antibiotic resistance to the beta-lactam antibiotics

Antibiotic resistance of both gram-positive and gram-negative bacteria is common, especially to the natural penicillins and earlier generation cephalosporins. The common (not all) mechanisms of antibiotic resistance are shown in the Table below.