John F. Prescott and Keith E. Baptiste Macrolides (macro meaning large and olide meaning lactone) are characterized by having a central 12‐ to 16‐membered lactone ring that has few or no double bonds and (apart from the azalides) no nitrogen atoms to which two or more sugar moieties are attached. The efficacy of this group of drugs against important human pathogens, including Campylobacter, Chlamydia, Legionella, and Mycobacterium species, has resulted in development of semisynthetic members with increased antibacterial activity, improved pharmacokinetics, and reduced adverse reactions. Long‐acting macrolides formulations have come into widespread use for the treatment and metaphylaxis of bovine and swine respiratory disease although antimicrobial resistance increasingly hinders their use. A different range of macrolides, with the exception of erythromycin, are approved for use in veterinary compared to human medicine, although there is regular extra‐label use of human‐approved macrolides such as azithromycin and clarithromycin in animals. Macrolides are classified according to the number of atoms comprising the lactone ring, e.g., 12‐, 13‐, 14‐, 15‐, or 16‐ (Figure 12.1). The 12‐member ring macrolides are no longer used in clinical practice. Tulathromycin, a semisynthetic macrolide approved for use in swine and cattle, consists of an equilibrated regioisomeric mixture of a 13‐membered ring (10%) and a 15‐membered ring (90%). The unique structural feature of this antimicrobial places it in a novel category of macrolides termed triamilides. The 14‐member ring group contains compounds of natural origin (erythromycin, oleandomycin) and semisynthetic derivatives (clarithromycin, roxithromycin, dirithromycin). The 15‐member ring is represented by azithromycin, gamithromycin, and one isomer of tulathromycin. The 15‐membered ring macrolides are termed azalides as they have a nitrogen atom in the lactone ring. The 16‐member group also contains both compounds of natural origin (spiramycin, josamycin, midecamycin) and semisynthetic derivatives (tilmicosin, tildipirosin). As a class, the macrolides exhibit broad distribution to tissues and, in the case of some of the newer drugs, prolonged half‐lives. Macrolides are most active against Gram‐positive pathogens, but the newer drugs also have good activity against Gram‐negative bacteria associated with respiratory disease in food animals, although resistance is increasing. Macrolides are also known for their exceptional intracellular accumulation within phagocytes. However, the precise pharmacodynamic relationships between intracellular concentrations and bacterial killing remain to be defined. Figure 12.1 Classification of macrolide antimicrobials according to the size of the macrocyclic lactone ring. Macrolides inhibit protein synthesis by reversibly binding to 50S subunits of the ribosome. They inhibit the transpeptidation and translocation process, causing premature detachment of incomplete polypeptide chains. They target the nascent peptide exit channel of the bacterial ribosome and have traditionally been thought of as plugging the channel. Recent evidence suggests that, rather than stopping global translation, macrolides selectively inhibit protein synthesis and modulate translation. Their binding sites on the 23S rRNA of the 50S ribosomal subunit overlap with those of lincosamides, streptogramins, ketolides, and oxazolidinones but are different from those of chloramphenicol. Macrolides are generally bacteriostatic drugs, but they may be bactericidal at high concentrations and against a low inoculum of some highly susceptible bacteria. Three different mechanisms account for most bacterial resistance to the action of macrolides: (1) rRNA methylation; (2) active efflux; and (3) enzymatic inactivation. rRNA methylation and active efflux are the mechanisms responsible in most resistant isolates. Many macrolide resistance genes are associated with mobile elements and thus have the capacity to spread between strains, species, and bacterial ecosystem. rRNA methylation, encoded by erythromycin‐resistant methylase (erm) genes, results in cross‐resistance to the macrolides, lincosamides, and streptogramin B (MLSB resistance). To date, at least 46 different rRNA methylases have been characterized. These methylase genes are widely distributed in both Gram‐positive and Gram‐negative bacteria and can be located on plasmids or transposons. The expression of erm genes can be constitutive or inducible. Inducible resistance occurs when enzyme induction is affected by exposure of the microorganism to 14‐ or 15‐member ring macrolides, but not to 16‐member ring macrolides. Efflux of macrolide antimicrobial agents is mediated by members of the ATP binding cassette family of proteins (msr genes) or by major facilitator superfamily transporters (mef genes). The msr genes are also associated with MLSB resistance. These proteins pump antimicrobial agents out of the cell or cellular membrane, thereby allowing the bacterial ribosomes to function again. Currently, at least 37 different efflux genes have been recognized. Some of these genes confer resistance to 14‐ and 15‐member ring macrolides while not interfering with susceptibility to 16‐member ring macrolides, ketolides, lincosamides, and streptogramin B. Other efflux genes lead to a variety of different resistance patterns, including resistance to all macrolides, lincosamides, and streptogramins. The efflux genes have been found in a variety of Gram‐positive and Gram‐negative bacteria. The third and least common mechanism of resistance is due to enzymatic inactivation. There are currently four esterase (ere genes) and 15 phosphorylase inactivating enzymes (mph genes) known to be involved in macrolide resistance. A proportion of macrolide‐resistant bacteria do not carry any of the known transferable macrolide resistance genes. These isolates typically have several mutations in their rRNA genes and/or ribosomal protein genes, modifications which confer macrolide resistance. The additive effect of these mutations accounts for the progressive reduction of susceptibility to macrolides, which is a common feature in many pathogens following prolonged use. As discussed in Chapter 24, under antimicrobial stewardship in food animals, macrolides were classified by the World Health Organization (WHO) as Highest Priority Critical Important Antimicrobials but the seventh edition is expected to reclassify them as Critically Important Antimicrobials. There have been earlier calls to revise the WHO classification (Trott et al., 2021). The 18 member macrolides (fidaxomicin) are expected to be classified as “human use only” in the seventh edition of the WHO list. As discussed in this chapter, resistance to macrolides in important bacterial pathogens of animals continues to rise almost inexorably, in the form of both reduced susceptibility and increased resistance, compounded by the spread of transferable macrolide resistance genes through mobile genetic elements, emphasizing the importance of the stewardship of this class of drugs. Although declining, tylosin is still used as a growth‐promoting feed additive in many countries globally (WHO, 2022), a practice that must stop. There have been relatively few studies investigating the interactions of macrolide antibiotics with other antimicrobial drugs. Combinations of erythromycin with other macrolides, lincosamides, and chloramphenicol are antagonistic in vitro. Erythromycin has been used alone or with an aminoglycoside to prevent or treat peritonitis after intestinal spillage, but it is not as effective as clindamycin or metronidazole in combination with an aminoglycoside. Combination of a macrolide and a fluoroquinolone or aminoglycoside may be synergistic, antagonistic, or indifferent depending on the microorganism studied. The combination of a macrolide with rifampin was originally described as synergistic in vitro against Rhodococcus equi, which spawned a tradition of recommending the oral administration of the combination for 40 years as the standard of treatment so as to reduce the mutation‐based emergence of resistance to each of these drugs (Giguere et al., 2011a). In vivo pharmacokinetic studies have shown a negative interaction of rifampin with clarithromycin or gamithromycin through altered pharmacokinetic parameters, including subtherapeutic concentrations of either clarithromycin or rifampin (Berlin et al., 2016, 2018). Rifampin has effects on the pregnane X receptor which regulates different enzymes of the cytochrome P450 complex, as well as efflux carrier proteins and transporter proteins (Chapter 18). Although clarithromycin or azithromycin combined with rifampin are effective treatments for R. equi pneumonia, no randomized controlled studies have confirmed the benefit of the combination over a macrolide alone (Giguère et al., 2011a). Combination of clarithromycin with doxycycline or minocycline has been showed to reduce the emergence of resistance in R. equi in vitro (Erol et al., 2021) and azithromycin and doxycycline to be as effective as azithromycin and rifampin for treatment of R. equi bronchopneumonia clinically (Wetzig et al., 2019). The combination of rifampin with a macrolide (especially clarithromycin) may have adverse effects that are perhaps best avoided by omission of rifampin but further study is required. Erythromycin and many other macrolides lead to inactivation of the cytochrome P450 enzyme complex. Thus, besides rifampin, concurrent administration of erythromycin increases concentrations of drugs that are primarily dependent upon CYP3A enzyme metabolism such as theophylline, midazolam, carbamazepine, omeprazole, and ranitidine. Clarithromycin and roxithromycin have lower affinity for the P450 system than erythromycin and other classic macrolides (except spiramycin). Azithromycin and spiramycin do not interact with the hepatic cytochrome P450 system and are not associated with the drug interactions observed with erythromycin and other macrolides. Macrolides have immunomodulatory effects that are beneficial for humans suffering from many inflammatory pulmonary diseases such as cystic fibrosis, idiopathic bronchiectasis, and chronic obstructive pulmonary disease. These effects are likely independent of the antibacterial activity of these drugs. Erythromycin, azithromycin, clarithromycin, and roxithromycin inhibit chemotaxis and infiltration of neutrophils into the airway and, subsequently, decrease mucus secretion. The mechanisms of action for the antiinflammatory properties of the macrolides are multifactorial and still under investigation (Mainguy‐Seers et al., 2022). Macrolides inhibit the production of many proinflammatory cytokines by suppressing the transcription factor nuclear factor‐kappa B or activator protein‐1. Macrolides also inhibit formation of leukotriene B4, which attracts neutrophils and inhibits superoxide anion release by neutrophils that may be present in the airway. In addition, macrolides block formation of adhesion molecules necessary for neutrophil migration. Recent studies suggest an effect of macrolides on both innate and adaptive immunity as well (Martinez‐Cortés et al., 2018). These antiinflammatory and immunomodulatory effects have been described in foals receiving erythromycin (Lakritz et al., 1997), and in cattle and pigs administered tilmicosin or tulathromycin (Fischer et al., 2011). Macrolides with 14‐ or 16‐member rings have prokinetic effects on the gastrointestinal tract by acting as motilin receptor agonists, producing gastrointestinal disturbances demonstrated in cattle, dogs, and horses. Erythromycins are produced as a complex of six components (A–F) by Saccharopolyspora erythraea (formerly Streptomyces erythraeus). Only erythromycin A has been developed for clinical use. Erythromycin has a macrocyclic lactone nucleus to which ketones and amino sugars are attached (Figure 12.2). Its base has a pKa of 8.8, is poorly soluble in water, and is unstable in gastric acid. Antimicrobial susceptibility categories given below are guidelines only; laboratory breakpoints available in veterinary medicine will vary with dosage, sites of infection, route of administration, and other variables (Chapter 2). Figure 12.2 Structural formulas of macrolides. Source: Courtesy of Jérôme del Castillo. The erythromycin base is highly susceptible to degradation from gastric acids. To circumvent this, orally administered erythromycin requires an enteric coating. However, this leads to considerable individual variation in absorption. Erythromycin is available for oral administration as the free base, the stearate or phosphate salts, and as estolate or ethylsuccinate esters. The stearate is hydrolyzed in the intestine to the active base, and the ethylsuccinate and estolate esters are absorbed as such and hydrolyzed in the body to the active base. Feeding interferes quite markedly with oral absorption. Like all macrolides, erythromycin is well distributed in the body, being concentrated in tissues, although penetration into cerebrospinal fluid is low. Prostatic fluid concentrations are approximately half of serum concentrations. The drug is metabolized and excreted largely in the bile and, although some intestinal reabsorption occurs, most is in feces. Urinary excretion is only 3–5% of the total administered. Erythromycin is available for parenteral injection as the base, glucoheptonate, or lactobionate. Parenteral administration causes tissue irritation at the site of administration. The incidence of serious adverse effects is relatively low and depends on the animal species. One problem shared with all macrolides is their irritating nature, which leads to severe pain on IM injection, thrombophlebitis and periphlebitis after IV injection, and an inflammatory reaction after intramammary administration. Dose‐related gastrointestinal disturbances result either from disruption of the normal intestinal microflora, or because of stimulatory effects on smooth muscle due to erythromycin binding to motilin receptors following oral administration. These adverse effects are not life threatening except in adult horses, where macrolides, because they are largely excreted in the bile, can lead to serious gastrointestinal adverse events (diarrhea, colic). Deaths have occurred due to Clostridioides difficile in adult horses administered erythromycin. Interestingly, severe C. difficile diarrheal illness has also developed in the mares of foals treated orally with erythromycin and rifampin for R. equi infection. This may be a direct effect of mares ingesting small quantities of antibiotic from the feces of their foals or an indirect effect of mares acquiring erythromycin‐resistant C. difficile infection from their foals, or a combination of these circumstances (Båverud et al., 1998). Deaths from typhlocolitis have also been reported in rabbits. Oral administration of erythromycin has caused severe diarrhea in ruminating calves. Because of this effect combined with poor absorption, oral administration of erythromycin to cattle and other herbivores is not recommended. The drug appears safe in dogs and cats. The estolate form has been associated with self‐limiting cholestatic hepatitis and jaundice with abdominal pain, especially with repeated and prolonged use or in patients with preexisting hepatic disease. Other adverse effects of erythromycin in foals include hyperthermia and respiratory distress that may be more marked in foals kept in high environmental temperatures, apparently the result of impaired sweat responses (Stieler et al., 2016). Dosages of erythromycin are shown in Table 12.1. When administered IV, erythromycin must be diluted and administered by slow infusion to prevent adverse reactions. Erythromycin is a drug of choice to prevent or treat Campylobacter jejuni diarrhea or abortion, or C. coli diarrhea, should treatment be required, although there are reports of resistance in isolates from different sources, including poultry (a major source of infection for humans) (van Vliet et al., 2022). Erythromycin is also used as an alternative to penicillin in penicillin‐allergic animals for the treatment of infections caused by susceptible Gram‐positive aerobes, a less useful alternative to clindamycin or metronidazole in anaerobic infections, an alternative to ampicillin or amoxicillin in the treatment of leptospirosis, and an alternative to tetracyclines in rickettsial infections. The generally bacteriostatic nature of the drug is a disadvantage of erythromycin and other macrolides, as is the pain on IM injection. Acquired resistance by bacteria for which erythromycin used to be recommended as a second‐choice drug increasingly limits its value without susceptibility testing. Erythromycin has limited use in respiratory disease, as H. somni, T. pyogenes, and anaerobic bacteria are often moderately susceptible, but some mycoplasma (M. bovis) and most Mannheimia haemolytica isolates are resistant. Due to the extreme pain associated with parenteral injection, it should be avoided when other antimicrobial drugs are available. Its use in these species has been mostly replaced by the newer macrolides discussed below. Erythromycin is perhaps most useful in its intramammary infusion form for lactating and dry‐cow therapy of mastitis where it has a short milk withdrawal time (36 hours). A single IM injection has been effective in the treatment of virulent footrot in sheep. Table 12.1 Usual dosages of selected macrolides in animals. a Cattle and sheep only. b Mainly indicated in foals. c Slow IV infusion. IM, intramuscular; IV, intravenous; PO, by mouth (per os); SC, subcutaneous. Erythromycin’s use in swine has been replaced by newer macrolides. Erythromycin is an alternative to penicillin G or trimethoprim‐sulfonamide in the treatment of staphylococcal and streptococcal infections. The potential for inducing severe diarrhea limits its use in adult horses. Erythromycin was commonly used in the oral treatment of R. equi pneumonia in foals, although azithromycin is both more convenient to administer and produces significantly greater concentrations in pulmonary epithelial lining fluid (PELF) and bronchalveolar lavage cells (Suarez‐Meier et al., 2007). Transferable erm‐based resistance, discussed below under Azithromycin, increasingly challenges its use. Intramuscular injection causes severe local irritation in horses. The combination of orally administered erythromycin and rifampin has been used successfully to treat experimentally induced Neorickettsia risticii infection and may represent an alternative to tetracyclines, although use in combination with rifampin should be avoided for the reasons discussed earlier. Erythromycin is a treatment of choice for Lawsonia intracellularis infections in foals (Lavoie et al., 2000). Erythromycin may be a second choice for infections caused by Gram‐positive cocci and anaerobic bacteria. It is the drug of choice in treating C. jejuni enteritis. A nation‐wide outbreak of extensively drug‐resistant, including erythromycin‐resistant, C. jejuni in puppies obtained from pet store puppies has been described in the United States (Francois Watkins et al., 2021), illustrating the importance of infection control as part of antimicrobial stewardship (Chapters 20, 24). Erythromycin is administered in water for the prevention and treatment of staphylococcal or streptococcal infection, necrotic dermatitis, infectious coryza, and M. gallisepticum infection. Tylosin is a macrolide antibiotic isolated from Streptomyces fradiae. Its chemical structure and its mechanism of action are similar to other macrolide antibiotics. Tylosin has a similar spectrum of activity to erythromycin. It is less active against bacteria but more active against a broad range of Mycoplasma spp. Resistance is discussed further below with long‐acting macrolides used to treat respiratory disease in cattle and swine since there is extensive cross‐resistance among macrolides. The pharmacokinetic properties of tylosin are characteristic of the macrolides in general. Tylosin is a weak base (pKa 7.1) and is highly lipid soluble. The elimination half‐life in dogs and cattle is about one hour with apparent volumes of distribution of 1.7 and 1.1 l/kg, respectively. The half‐life is considerably longer in sheep, goats, and pigs, at approximately four hours. Tylosin is a relatively safe drug. Its toxic effects are generally similar to those reported for erythromycin. The drug is irritating to tissue when administered IM or SC. Pigs have been reported to react to injection by developing edema, pruritus, edema of rectal mucosa, and mild anal protrusion. These effects may in part be attributed to the drug vehicle. Tylosin has been reported to cause fatal diarrhea in a horse. Inadvertent feeding of dairy cows with a concentrate contaminated with 7–20 ppm of tylosin resulted in ruminal stasis, inappetence, foul‐smelling feces, and decreased milk production. Many of the cows became hyperesthetic and some became recumbent. Intravenous administration in cattle has produced shock, dyspnea, and depression. Tylosin and spiramycin have induced contact dermatitis in veterinarians. Tylosin is primarily used in veterinary medicine. It is administered by IM injection (Table 12.1), by the intramammary route, or for feed incorporation in swine, cattle and poultry. Tylosin tartrate is readily absorbed from the intestine, but tylosin phosphate is relatively poorly absorbed. Tylosin was at one time used extensively as a growth‐promoting feed additive but its continued use for such a purpose is wrong. Tylosin is not as active as erythromycin against most bacteria, but has greater activity against Mycoplasma spp. in swine, although it has been replaced by the more active tiamulin and in cattle by the newer macrolides. Swine Brachyspira spp. are now all largely resistant (Hampson et al., 2019). Apart from its use against some Mycoplasma, tylosin is, like erythromycin, a second‐choice antibiotic in most clinical situations. Tylosin has been used in cattle in the past to treat pneumonia associated with Mycoplasma bovis, bovine respiratory disease caused by Mannheimia, Pasteurella multocida, and Histophilus somni, and otitis media and interna in calves but its use for these and other purposes has been largely replaced by newer macrolides. Other possible indications include treatment of footrot, metritis, pinkeye, and mastitis caused by Gram‐positive cocci. The susceptibility of M. bovis to tylosin has reduced markedly over time (Cai et al., 2018), and clearly resistant strains have emerged (Jelinksi et al., 2020) in Canada and elsewhere (Liu et al., 2020). Such resistance is associated with ribosomal mutations (Bokma et al., 2021). In cattle, tylosin has been successful in controlling and eliminating experimental Mycoplasma mycoides pneumonia. In calves, the drug has been used effectively to treat M. bovis pneumonia and arthritis. In goats, tylosin was a drug of choice for treating Mycoplasma pneumonia, such as that caused by M. mycoides spp. capri. A high dosage of 25–35 mg/kg IV at 8–12‐hour intervals is recommended. In feedlot cattle, in‐feed tylosin is used extensively in North America in the prevention of liver abscesses in animals predisposed to infection from high‐grain diets that cause low‐grade rumenitis. The concern about such extensive and long‐term use is that resistance may develop in the bacterial microbiota which may impact human as well as animal health. Studies of the incidence and antimicrobial resistance of different Enterococcus species from humans and beef cattle and their environments not surprisingly showed distinct species and resistance diversity (niche specificity) (Zaheer et al., 2020). Macrolide (ermB) resistance was prevalent in all enterococci. However, one study showed minimal impact on antimicrobial resistance in selected members of the microbiota (Schmidt et al., 2020). There is little recent information on the prevalence of resistance of Fusobacterium necrophorum and Trueperella pyogenes from liver abscesses in feedlot cattle. Concern about resistance and the inappropriate long‐term prophylactic use of antimicrobial drugs (Cazer et al., 2020) has led to ongoing promising studies on the effect of intermittent or a reduced time of administration of tylosin on development of liver abscesses (Müller et al., 2018; Davedow et al., 2020). In swine, tylosin has been used for treatment of atrophic rhinitis, bacterial pneumonia, erysipelas, leptospirosis, and proliferative enteropathy, but has largely been replaced by more active macrolides or pleuromutulins. Mycoplasma hyorhinis isolated recently in several European countries (Klein et al., 2022) show frequent acquired resistance although M. hyopneumoniae and M. hyosynoviae are susceptible (Rosales et al., 2020; de Jong et al., 2021a). Tylosin resistance is extensive in Brachyspira hyodysenteriae and B. pilosicoli (Hampson et al., 2019; Arnold et al., 2022). Resistance in Streptococcus suis is extensive. Injection of tylosin has been fatal to horses. There is no experience with its oral administration but no indication for such use has been identified, which likely could result in severe enterocolitis. Tylosin has been used successfully in dogs to treat abscesses, wound infections, tonsillitis, tracheobronchitis, and pneumonia caused by pathogens such as staphylococci, streptococci, anaerobes, and Mycoplasma. Tylosin is often effective in the treatment of the upper respiratory tract infection complex of cats, possibly because of its effect against Chlamydia and Mycoplasma. Tylosin administered orally has been used in the treatment of Staphylococcus pseudintermedius pyoderma in dogs, but its use may be limited by resistance. It is a drug of choice for treatment of Campylobacter jejuni diarrhea in dogs.
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Macrolides, Azalides, and Ketolides
Mechanism of Action
Resistance
Public Health Considerations of Resistance
Drug Interactions
Antiinflammatory and Prokinetic Activities of Macrolides
Macrolides Approved for Veterinary Use: Erythromycin, Tylosin, Spiramycin, Tilmicosin, Tulathromycin, Gamithromycin, Tildipirosin, and Tylvalosin
Erythromycin
Antimicrobial Activity
Pharmacokinetic Properties
Toxicity and Adverse Effects
Administration and Dosage
Clinical Applications
Cattle, Sheep, and Goats
Species
Drug
Dosage (mg/kg)
Route
Interval (h)
Dog/cat
Erythromycin
10–20
PO
8–12
Clarithromycin
5–10
PO
12
Azithromycin
5 (cat), 10 (dog)
PO
24
Tylosin
10–20
PO
12
5–10
IM
12
Ruminants
Erythromycin
1.1–2.2
IM
24
Tylosin
4–10
IM
24
Tilmicosina
10
SC
Single dose
Tulathromycin
2.5
SC
Single dose
Gamithromycin
6
SC
Single dose
Tildipirosin
4
SC
Single dose
Horsesb
Erythromycin
25
PO
6–8
Erythromycin
5
IVc
6
Clarithromycin
7.5
PO
12
Azithromycin
10
PO, IVc
24–48
Swine
Erythromycin
2–20
IM
12–24
Tylosin
9
IM
12–24
Tilmicosin
200–400 g/ton of feed
Tulathromycin
2.5
IM
Single dose
Tildipirosin
4
IM
Single dose
Tylvalosin
50–100 g/ton of feed
Tylvalosin
50 ppm
Water
Swine
Horses
Dogs and Cats
Poultry
Tylosin
Antimicrobial Activity
Pharmacokinetic Properties
Toxicity and Adverse Effects
Administration and Dosage
Clinical Applications
Cattle, Sheep, and Goats
Swine
Horses
Dogs and Cats
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