Chemistry

The penicillins, cephalosporins, carbapenems, monobactams, and penems are referred to as beta‐lactam antibiotics. Rupture of the beta‐lactam ring, which is brought about enzymatically by bacterial beta‐lactamases, results in loss of antibacterial activity. Hypersensitivity reactions appear to be associated with the active moieties of the beta‐lactam drugs, and although the risk is small, caution should be exercised when administering cephalosporins to penicillin‐sensitive animals because these drugs are of similar structure. In drug development, substitutions can be made on the beta‐lactam ring for specific purposes, such as increasing resistance to beta‐lactamases of clinically important families or species of bacteria; enhancing activity against selected pathogens; or ensuring favorable pharmacokinetic properties. Thus, some semisynthetic beta‐lactam drugs have been designed for specific purposes.

Mechanism of Action

Beta‐lactam antibiotics prevent the bacterial cell wall from forming by interfering with the final stage of peptidoglycan synthesis. They inhibit the activity of the transpeptidase and other peptidoglycan‐active enzymes called penicillin‐binding proteins (PBPs) (transpeptidases, carboxypeptidases), which catalyze cross‐linkage of the glycopeptide polymer units that form the cell wall. The PBPs are further classified according to their molecular mass into high molecular weight PBPs and low molecular weight PBPs. In the presence of beta‐lactam drugs, the PBPs are inactivated when covalently bound to the beta‐lactam ring. The drugs exert a bactericidal action but cause lysis only of growing cells, that is, cells which are undergoing active cell wall synthesis. Also, the change in the bacterial cell wall attracts phagocytic activity by immune cells. In Gram‐positive bacteria the beta‐lactams not only prevent final peptidoglycan cross‐linking, but also stimulate lipoteichoic acid release, causing a suicide response by degradation of peptidoglycan by autolysins.

Variation in the activity of different beta‐lactams results, in part, from differences in affinity of the molecules for the PBPs. The difference in susceptibility between Gram‐positive and Gram‐negative bacteria depends on differences in receptor sites (PBPs), on the relative amount of peptidoglycan present (Gram‐positive bacteria possess far more), on the ability of the drugs to penetrate the outer cell membrane of Gram‐negative bacteria, and on resistance to the different types of beta‐lactamase enzymes produced by the bacteria. These differences are summarized in Figures 7.3 and 7.4.

Beta‐lactam antibiotics are bactericidal drugs with slower kill rates than those exhibited by aminoglycosides or fluoroquinolones. Killing activity starts after a lag period. Penicillins tend to be slightly more active in a slightly acidic environment (pH 5.5–6.5), perhaps because of enhanced membrane penetration. Against Gram‐positive bacteria, all beta‐lactams exhibit an in vitro postantibiotic effect. This does not carry over for the streptococci in vivo but does for strains of staphylococci that are susceptible to penicillins. The beta‐lactams do not exhibit a postantibiotic effect against Gram‐negative bacteria, with the possible exception of carbapenems against Pseudomonas. Optimal antibacterial efficacy is time and not concentration dependent (Chapter 5) and therefore requires that serum concentrations exceed the MIC of the pathogen for essentially the entire dosing interval for bactericidal effects. These drugs are best administered frequently or by continuous infusion.

Resistance to Beta‐lactam Antibiotics

Changes to PBPs can confer resistance. A change in the number of PBPs impacts the amount of drug that can bind to that target. This can occur by an increase in PBPs that have a decrease in drug‐binding ability or decrease in PBPs with normal drug binding. A change in structure (e.g., PBP2a in S. aureus by acquisition of the mecA gene) may decrease the ability of the drug to bind, or totally inhibit drug binding. The level of resistance is determined by how many and to what extent targets are modified.

In Gram‐positive bacteria, especially S. aureus, resistance to penicillin G is mainly through the production of beta‐lactamase enzymes that break the beta‐lactam ring of most penicillins. Staphylococcus spp. secretes beta‐lactamase enzymes extracellularly as inducible exoenzymes that are plasmid mediated (Figure 7.3). Inherent resistance to penicillin G of many Gram‐negative bacteria results from low permeability of the Gram‐negative cell wall, lack of PBPs, and a wide variety of beta‐lactamase enzymes (Figure 7.4). Most Gram‐negative bacteria inherently express low levels of species‐specific, chromosomally mediated beta‐lactamase enzymes within the periplasmic space, which sometimes contribute to resistance. These enzymes hydrolyze susceptible cephalosporins more rapidly than penicillin G, but they poorly hydrolyze ampicillin, carbenicillin, and beta‐lactamase‐resistant penicillins.

Production of plasmid‐mediated beta‐lactamases is widespread among common Gram‐negative bacterial pathogens. The enzymes are constitutively expressed, present in the periplasmic space, and cause high‐level resistance. The majority are penicillinases rather than cephalosporinases (Figure 7.4). The most widespread are those classified based on their hydrolytic activity as TEM‐type beta‐lactamases, which readily hydrolyze penicillin G and ampicillin rather than methicillin, cloxacillin, or carbenicillin. The less widespread OXA‐type beta‐lactamases hydrolyze penicillinase‐stable penicillins (oxacillin, cloxacillin, and related drugs). More details on beta‐lactamases are given in Chapter 9. Beta‐lactamases probably evolved from PBPs as a protective mechanism for soil organisms exposed to beta‐lactams in nature. Because of the spread of transferable resistance, beta‐lactamase production by pathogens is now widespread and extensive.

A major advance has been the discovery of broad‐spectrum beta‐lactamase‐inhibitory drugs (e.g., clavulanic acid, sulbactam, tazobactam). These drugs have weak antibacterial activity but show extraordinary synergism when administered with penicillin G, ampicillin (or amoxicillin), and ticarcillin, because of the irreversible binding of the beta‐lactamase enzymes of resistant bacteria. Other beta‐lactamase inhibitors, such as cefotaxime and carbapenems, have potent antibacterial activity in their own right (Chapter 9).

General Considerations

The acidic radical (R), attached to the amino group of 6‐aminopenicillanic acid (Figure 7.1) determines the susceptibility of the resulting penicillin to hydrolytic degradation or enzymatic inactivation by bacterial beta‐lactamase, and the antibacterial activity of the molecule. Both these factors influence the clinical effectiveness of penicillins, which is also determined by the concentration attained at the site of infection. The nature of the acidic radical has little influence on the rate of elimination of penicillins but determines the extent of plasma albumin binding and, to a lesser degree, membrane‐penetrating ability. The 6‐aminopenicillanic acid moiety and structure of the acid radicals of some penicillins are shown in Figure 7.5.

Penam penicillins are readily distinguished on the basis of antimicrobial activity into six groups (“generations”), which largely correspond to their time of introduction into clinical use (Table 7.1): (1) benzyl penicillin and its long‐acting parenteral forms; (2) orally absorbed penicillins similar to benzyl penicillin; (3) staphylococcal penicillinase‐resistant isoxazolyl penicillins; (4) extended‐ or broad‐spectrum penicillins; (5) antipseudomonal penicillins; (6) beta‐lactamase resistant penicillins.

Since the 1940s, there has been progressive development of penicillins for clinical use, resulting in derivatives with similar activity to benzyl penicillin, but which can be administered orally and/or are resistant to S. aureus beta‐lactamase (penicillinase). Subsequently, orally administered penicillins were developed with a broader spectrum of activity, which involved greater Gram‐negative antibacterial activity, and penicillins active against P. aeruginosa. Despite considerable effort at identifying beta‐lactamase resistant penam penicillins, with the exception of temocillin, extended‐spectrum penicillins are susceptible to beta‐lactamase‐producing Gram‐negative bacteria. For this reason, the use of penicillins against common Gram‐negative bacteria is limited in favor of more recently introduced cephalosporin beta‐lactams (Chapter 8) or combination with beta‐lactamase inhibitors (Chapter 9).

Mechanism of Action

The targets of all beta‐lactam drugs are the PBPs found on the outside of the cytoplasmic membrane, which are involved in synthesizing and remodeling the cell wall. Susceptibility of a bacterium to a penicillin depends on a combination of affinity for the PBP, ability to penetrate the cell wall, and ability to resist beta‐lactamase enzymes (Figure 7.3, 7.4). There are usually 4–7 PBPs present in the bacterial cell wall that are the targets for penicillins. The bactericidal effect in Gram‐negative bacteria results from osmotically induced lysis of cells weakened by loss of their peptidoglycan layer. Gram‐positive bacteria have considerably greater quantities of peptidoglycan in their cell wall than Gram‐negative bacteria and an effect of beta‐lactams is to prevent the final peptidoglycan cross‐linking, which gives peptidoglycan its strength. Also, beta‐lactams cause the release of lipoteichoic acid, leading to a suicide response by degradation of peptidoglycan by autolysins (endogenous endopeptidase, carboxypeptidase PBPs).

For some Gram‐positive cocci, exposure to beta‐lactam antibiotics, above an optimal killing concentration, results in a reduction of killing, which can be considerable (the “Eagle” or paradoxical effect). Its basis appears to be interference of growth by penicillin binding to PBPs other than the major target PBP. Since beta‐lactams are effective only against growing, actively cell wall‐synthesizing bacteria, failure to grow results in failure to be killed. The Eagle effect is an important concept, since there may be a tendency to overdose with beta‐lactam antibiotics, because they are generally nontoxic.

Antimicrobial Activity

Benzyl penicillin, penethamate hydriodide, and orally administered benzyl penicillins (phenoxymethyl penicillin) have outstanding activity against many susceptible Gram‐positive bacteria, notably beta‐hemolytic streptococci, nonresistant staphylococci, Actinomyces spp., Arcanobacterium spp., Bacillus spp., Clostridium spp., Corynebacterium spp., and Erysipelothrix rhusiopathiae. Susceptible Gram‐negative species include some Bacteroides spp., some Fusobacterium spp., and a variety of Gram‐negative aerobic bacteria such as Haemophilus spp., and many Pasteurella spp. Enterobacterales, Bacteroides fragilis, most Campylobacter spp., Nocardia spp. and Pseudomonas spp. are resistant.

Penicillinase‐resistant, antistaphylococcal isoxazolyl penicillins (cloxacillin, dicloxacillin, methicillin, nafcillin, oxacillin) have activity similar to but slightly less than that of benzyl penicillin, with the exception that they are active against penicillinase‐producing S. aureus. Extended‐spectrum penicillins (aminobenzylpenicillins such as ampicillin and its esters, and amoxicillin) retain the activity of benzyl penicillin against Gram‐positive bacteria but have increased activity against Gram‐negative bacteria including E. coli, Proteus spp., and Salmonella spp. They are, however, ineffective against P. aeruginosa and are inactivated by beta‐lactamases. Mecillinam, another member of the extended penicillin group, differs from aminobenzylpenicillins in its lower activity against Gram‐positive bacteria but considerably greater activity against Gram‐negative bacteria, including a broad spectrum of the Enterobacterales, although it is still inactivated by many beta‐lactamases. Penicillins (carboxypenicillins, ureidopenicillins) active against P. aeruginosa (carbenicillin, azlocillin, mezlocillin, piperacillin) are effective against both Gram‐positive and Gram‐negative bacteria, including P. aeruginosa.

It is important to note that acquired antimicrobial resistance is emerging globally but varies greatly between countries. For example, rates of penicillin resistance to staphylococci vary in different countries and even within different species in the same country (e.g., horses and dogs). Veterinarians should consult local antibiograms (where available) to assist with empirical prescribing decisions. Beta‐haemolytic streptococci species remain highly susceptible to penicillin universally. Although resistance has been described in S. equi subsp. equi isolated from the upper respiratory tract in horses in the United Kingdom (Fonseca et al., 2020), these isolates did not undergo repeated susceptibility testing or further analysis to confirm this finding, which should be performed in all cases where penicillin resistance in beta‐haemolytic streptococci species is detected.

Resistance to Penam Penicillins

Most resistance results from production of a beta‐lactamase enzyme, although modification of PBPs with reduced drug affinity or reduced bacterial permeability are additional and sometimes concurrent mechanisms of intrinsic or acquired resistance to penam penicillins. Efflux mechanisms and modification of porins in Gram‐negative bacteria that prevent entry of penicillins are also recognized. Beta‐lactamases are discussed in Chapter 9. Resistance because of exogenously produced beta‐lactamase is now widespread in S. aureus, particularly in clinical isolates, because of mobile genetic elements.

Among Gram‐negative bacteria, plasmids encoding beta‐lactamases have also become widespread and are the cause of extensive acquired resistance. Modification of PBPs is recognized to be increasingly important as another mechanism of resistance to penam penicillins, particularly among Gram‐positive organisms. For example, a mutation in PBP5 in Enterococcus faecalis causes loss of affinity and beta‐lactam resistance. A third mechanism is to prevent the beta‐lactam antibiotic from reaching the target by altering the permeability of the outer membrane or increasing efflux pump activity. This is one of the main causes of resistance in Pseudomonas aeruginosa and other pathogenic Gram‐negative bacteria.

The most important type of penam penicillin resistance in human medicine is methicillin (oxacillin) resistance in S. aureus (MRSA), which is widespread in humans in some countries, notably Japan and the United States. Also, methicillin resistance is well established in animal populations, notably in dogs, horses, and swine, and appears to reflect the incidence of infection in humans from whom these strains were acquired (Price et al., 2012; Chen and Wu, 2020). Methicillin resistance in S. aureus causing bovine mastitis varies by region, with the highest prevalence in Asia (6.5%) and the lowest in Europe (1.2%), but prevalence appears to be increasing (Zaatout and Hezil, 2022).

The reason(s) for the emergence of MRSA in animals since 2000, and of livestock‐associated (LA) MRSA infections, are still unclear but represent host adaptation of particular clonal types to livestock, with antimicrobial resistance developing through selection by antimicrobial use. LA‐MRSA has been spreading rapidly among pig herds in concurrence with the common use of high‐dose zinc oxide veterinary medicinal products, used to prevent postweaning diarrhea in piglets. The gene coding for zinc and cadmium resistance is located within the same mobile genetic element as a certain type of Mec, which is a precursor for the methicillin resistance gene mecA, conferring resistance to broad‐spectrum β‐lactams. In addition, animal MRSA strains are often hospital associated and can contaminate veterinary hospital environments, including hospital personnel, to a remarkable extent. Human subclinical and even clinical infections have been acquired from animal sources. MRSA are regarded as resistant to all beta‐lactam antimicrobials and are commonly, but not always, resistant to other antimicrobials. Methicillin‐resistant S. pseudintermedius (MRSP) is also increasingly isolated from dogs and cats and, like MRSA, is regarded as resistant to all beta‐lactam antibiotics. Emergence of new and diverse lineages is well documented (Phophi et al., 2023). They commonly also have other multidrug resistances.

Methicillin resistance is more frequent in coagulase‐negative Staphylococcus spp. Coagulase‐negative Staphylococcus spp. have rarely been associated with clinical disease in the past but may be increasing in significance in companion animal practice where Staphylococcus schleiferi appears to be a significant pathogen in some cases of canine otitis externa (Lee et al., 2019).

Pharmacokinetic Properties

The penicillins are organic acids that are generally available as the sodium or potassium salt of the free acid. In dry, crystalline form, penicillins are stable but lose their activity rapidly when dissolved. Apart from the isoxazolyl penicillins (cloxacillin, dicloxacillin, oxacillin) and penicillin V, acid hydrolysis in the stomach limits the systemic availability of most penicillins from oral preparations.

The penicillins (pK_a 2.7) are predominantly ionized in plasma, have relatively small apparent volumes of distribution (0.2–0.3 l/kg), and have short half‐lives (0.5–1.2 hours) in all species of domestic animals. After absorption, they are widely distributed in the extracellular fluids of the body, but cross biological membranes poorly since they are ionized and poorly lipid soluble. Concentration in milk, for example, is about one‐fifth that of serum. Entry across biological membranes or through the blood–brain or blood–cerebrospinal fluid barrier is enhanced by inflammation, so that inhibitory drug concentrations may be attained at these sites that are normally inaccessible to penicillin.

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