Chemistry

The aminoglycoside antibiotics – streptomycin, dihydrostreptomycin, kanamycin, gentamicin, tobramycin, amikacin, and neomycin – are large molecules with numerous amino acid groups, making them basic polycations that are highly ionized at physiological pHs. Their polarity largely accounts for the pharmacokinetic properties that are shared by all members of the group. Chemically, they consist of a hexose nucleus to which amino sugars are attached by glycosidic linkages. This is why these molecules are also referred to as aminocyclitols or aminoglycosidic aminocyclitols.

The aminoglycosides can be divided into four groups on the basis of the type and substitution pattern of their aminocyclitol molecule: derivatives containing the aminocyclitol streptidine (e.g., streptomycin and dihydrostreptomycin), derivatives containing the aminocyclitol streptamine (e.g., spectinomycin), derivatives containing a 4,5‐disubstituted deoxystreptamine moiety (e.g., neomycin), and derivatives containing a 4,6‐disubstituted deoxystreptamine moiety (e.g., gentamicin, kanamycin, amikacin, tobramycin).

Mechanism of Action

Aminoglycosides must penetrate the bacteria to assert their effect. Penetration can be enhanced by the presence of a drug that interferes with cell wall synthesis, such as a beta‐lactam antibiotic. Susceptible, aerobic Gram‐negative bacteria actively pump the aminoglycoside into the cell. This is initiated by an oxygen‐dependent interaction between the antibiotic cations and the negatively charged ions of the bacterial membrane lipopolysaccharides. This interaction displaces divalent cations (Ca⁺⁺, Mg⁺⁺), which affects membrane permeability. Once inside the bacterial cell, aminoglycosides bind to the 30S ribosomal subunit and cause a misreading of the genetic code, interrupting normal bacterial protein synthesis. This leads to changes in the cell membrane permeability, resulting in additional antibiotic uptake, further cell disruption, and, ultimately, cell death.

The extent and types of misreading vary because different members of the group interact with different proteins. Streptomycin acts at a single site but the other drugs act at several sites. Other effects of aminoglycosides include interference with the cellular electron transport system, induction of RNA breakdown, inhibition of translation, interference with DNA metabolism, and damage to cell membranes. The bactericidal effect is through the formation of abnormal cell membrane channels by misread proteins.

Aminoglycoside action is bactericidal and dose (concentration) dependent. For example, gentamicin concentrations in the range of 0.5–5.0 μg/ml are bactericidal for Gram‐positive and some Gram‐negative bacteria. At 10–15 μg/ml, gentamicin is effective against the more resistant bacteria such as Pseudomonas aeruginosa, Klebsiella pneumoniae, and Proteus mirabilis. The clinical implication is that high initial doses increase ionic bonding, which enhances the initial concentration‐dependent phase of rapid antibiotic internalization and leads to greater immediate bactericidal activity. Human clinical studies demonstrate that proper initial therapeutic doses of aminoglycosides are critical in reducing mortality from Gram‐negative septicemia.

For antimicrobials whose efficacy is concentration dependent, high plasma concentration levels relative to the MIC of the pathogen (Cmax:MIC ratio, also known as the inhibitory quotient or IQ) and the area under the plasma concentration‐time curve that is above the bacterial MIC during the dosage interval (area under the inhibitory curve, AUIC = AUC/MIC) are the major determinants of clinical efficacy. For the aminoglycosides, a Cmax:MIC ratio of 10 was suggested to achieve optimal efficacy (McKellar et al., 2004). Recent work suggests that the area under the plasma concentration–time curve (AUC)/MIC ratio may be a more reliable indicator of bacterial killing and clinical efficacy (Bland et al., 2018). This human study suggests that an AUC/MIC ratio of 30–50 for aminoglycoside therapy may be effective for noncritically ill/immunocompetent patients with less serious Gram‐negative infections such as urinary tract infections. An AUC/MIC target of 80–100 may be prudent when treating patients with aminoglycoside monotherapy or in critically ill patients with serious Gram‐negative infections such as nosocomial pneumonia. Further evidence is needed in veterinary patients to determine the efficacy and safety of this PK/PD approach.

The aminoglycosides have a significant postantibiotic effect (PAE) – the period of time where antimicrobial concentrations are below the bacterial MIC but the antimicrobial‐damaged bacteria are more susceptible to host defenses. The duration of the PAE tends to increase as the initial aminoglycoside concentration increases.

Antimicrobial Activity

The antimicrobial action of the aminoglycosides is directed primarily against aerobic, Gram‐negative bacteria. Because bacterial uptake is oxygen dependent, they are not active against facultative anaerobes or aerobic bacteria under anaerobic conditions. They are less potent in hyperosmolar environments or low pH environments. Purulent debris at the infection site binds ionically to aminoglycosides and inactivates them. They are active against some Gram‐positive bacteria, such as Staphylococcus spp. Emerging strains of methicillin‐resistant Staphylococcus aureus (MRSA) and Staphylococcus pseudintermedius (MRSP) typically retain susceptibility to gentamicin and/or amikacin. Enterococci are inherently resistant (Chapter 2), and therapy against streptococci is more effective when combined with a beta‐lactam antibiotic. Salmonella and Brucella spp. are intracellular pathogens and are often resistant. Some mycobacteria, spirochetes, and mycoplasma are susceptible.

In potency, spectrum of activity, and stability to enzymes from plasmid‐mediated resistance, amikacin > tobramycin ≥ gentamicin > neomycin = kanamycin > streptomycin. Amikacin was developed from kanamycin and has the broadest spectrum of activity of the aminoglycosides. It is effective against Gram‐negative strains not susceptible to other aminoglycosides because it is more resistant to bacterial enzymatic inactivation. It is also considered the least nephrotoxic, but it is less efficacious against streptococci than gentamicin. Streptomycin and dihydrostreptomycin are the most active of these drugs against mycobacteria and Leptospira and the least active against other organisms. Paromomycin is effective against some protozoa and cestodes and is the only aminoglycoside with clinically important antileishmanial activity.

The bactericidal action of the aminoglycosides on aerobic Gram‐negative bacteria is markedly influenced by pH, being most active in an alkaline environment. Increased local acidity secondary to tissue damage or bacterial destruction may explain the failure of aminoglycosides to kill usually susceptible pathogens. Another factor affecting activity is the presence of purulent debris, which ionically binds to aminoglycosides and inactivates them. When using an aminoglycoside to treat purulent infections (e.g., abscesses), surgical debridement and/or drainage increases efficacy.

Resistance to Aminoglycoside Antibiotics

The most common mechanism of bacterial resistance to aminoglycosides is chemical modification by aminoglycoside‐modifying enzymes (AMEs) (Garneau‐Tsodikova and Labby, 2016). The AMEs originally had roles in normal cellular metabolism but have evolved to modify aminoglycosides with selective pressure from exposure to these antibiotics. The AMEs consist of three subclasses: aminoglycoside N‐acetyltransferases (AACs), aminoglycoside O‐nucleotidyltransferases (ANTs), and aminoglycoside O‐phosphotransferases (APHs). Each AME modifies an aminoglycoside at a specific position, such as an exposed hydroxyl or amino groups to prevent ribosomal binding, and this information is included in the enzyme name. Bifunctional enzymes exist and are capable of multiple types of aminoglycoside modification. AAC(6′)‐Ib is the most prevalent and clinically relevant AME, with approximately 50 variants of AAC(6′)‐Ib in numerous Gram‐negative bacterial species. The AMEs are present in the periplasmic space of bacteria, so that extracellular inactivation of drug does not occur.

The genes for AMEs are highly mobile; they are transferred on plasmids, integrons, transposons, and other transposable gene elements, often along with other resistance genes (such as beta‐lactamases). A single type of plasmid may confer cross‐resistance to multiple aminoglycosides and to other unrelated antimicrobials. A single bacterial isolate may have any one of a variety of combinations of resistance to different antibiotics conferred by the particular plasmid it carries. For example, one E. coli strain may be simultaneously resistant to ampicillin, apramycin, chloramphenicol, gentamicin, kanamycin, sulfonamide, streptomycin, tetracycline, and trimethoprim.

Antimicrobial resistance in organisms such as E. coli and Salmonella species is a focus of international research due to potential transference of antimicrobial resistance from animal to human pathogens. Several strategies are being investigated to overcome resistance caused by AMEs, including regulating AME expression, designing new aminoglycosides that evade AMEs, and designing of AME inhibitors.

Aminoglycosides target the A‐site of the bacterial ribosome to disrupt protein translation. Other acquired mechanisms of resistance include mutations of the ribosome or enzymatic modifications of the ribosome. The bacterial A‐site is located on the 16S RNA of the 30S bacterial ribosomal subunit. One known mechanism of aminoglycoside resistance occurs from mutations in the rrs gene, which codes for 16S rRNA, that hinders aminoglycoside binding. The aminoglycoside binding site may also be modified enzymatically by 16S ribosomal RNA methyltransferases (RMTases). The RMTases naturally occur in actinomycetes, the bacteria from which aminoglycosides were originally isolated. The RMTases are acquired by other bacteria by uptake of a plasmid containing the RMTase gene. The clinical prevalence of RMTases is low but increasing. This is a considerable threat to human and veterinary medicine because RMTases confer resistance to many clinically relevant aminoglycosides, including amikacin.

In order to have their bactericidal activity, aminoglycosides must cross the bacterial cell wall. The structure of the cell wall of Gram‐negative bacteria confers innate resistance to aminoglycosides. The outward facing half of the outer membrane of Gram‐negative bacteria consists of sugar‐functionalized lipopolysaccharides (LPSs), with a net negative charge, attracting cationic aminoglycosides. The most common LPS modification leading to reduced aminoglycoside uptake is the incorporation of the positively charged 4‐amino‐4‐deoxy‐L‐arabinose sugar, which effectively reduces the net negative charge of the LPS layer, decreasing affinity for aminoglycosides. Bacterial strains with reduced cell wall permeability and consequently 2–4‐fold increases in MIC may be selected during treatment with aminoglycosides. Such strains show cross‐resistance to all other drugs within the group.

If the aminoglycoside manages to penetrate the bacterial cell wall, active expulsion by efflux pumps may prevent effective intracellular concentrations. There are five families of efflux systems: the major facilitator superfamily, the ATP‐binding cassette family, the resistance‐nodulation division family (RND), the small MDR family, and the multidrug and toxic compound extrusion family. The majority of genes for efflux mechanisms are located on the chromosome, but members of the major facilitator superfamily are also located on plasmids. While the contribution of efflux pumps to aminoglycoside resistance is low overall, efflux pump expression may be used to monitor resistance to other classes of antimicrobials as a biomarker for determining how bacteria become multidrug resistant (Swick et al., 2011).

Chromosomal mutation resulting in resistance is relatively unimportant except for streptomycin and dihydrostreptomycin, where it occurs readily as a result of a single‐step mutation to high‐level resistance. For the other drugs, chromosomal resistance develops slowly, because there are many 30S ribosomal binding sites.

Both subinhibitory and inhibitory aminoglycoside concentrations produce resistance in bacterial cells surviving the initial ionic binding. This first‐exposure adaptive resistance is due to upregulation of aminoglycoside efflux pumps (Sidhu et al., 2012). Exposure to one dose of an aminoglycoside is sufficient to produce resistant variants of an organism with altered metabolism and impaired aminoglycoside uptake. In vitro, animal and clinical studies show that the resistance occurs within 1–2 hours of the first dose. The duration of adaptive resistance relates directly to the half‐life of elimination of the aminoglycoside. With normal aminoglycoside pharmacokinetics, the resistance may be maximal for up to 16 hours after a single dose, followed by partial return of bacterial susceptibility at 24 hours and complete recovery at 40 hours (Barclay and Begg, 2001). The clinical significance of this phenomenon is that frequent dosing or constant infusion of an aminoglycoside is less effective than high‐dose, once‐daily dosing.

Pharmacokinetic Properties

Aminoglycosides are poorly absorbed from the normal gastrointestinal tract, but are well absorbed after IM or SC injection. Following parenteral administration, effective concentrations are obtained in synovial, perilymph, pleural, peritoneal, and pericardial fluid. When given to neonates or animals with enteritis, oral absorption may be significantly increased and result in violative tissue residues in food animals. When given by intrauterine or intramammary infusion to cows, gentamicin is well absorbed and results in prolonged tissue residues.

Aminoglycosides bind to a low extent to plasma proteins (less than 25%). As they are large molecules and highly ionized at physiological pH values, they are poorly lipid soluble and have limited capacity to enter cells and penetrate cellular barriers. These drugs do not readily attain therapeutic concentrations in transcellular fluids, particularly cerebrospinal and ocular fluid. The milk‐to‐plasma equilibrium concentration ratio is approximately 0.5. Their apparent volumes of distribution are relatively small (<0.35 l/kg) and their plasma elimination half‐lives are short (1–2 hours) in domestic animals. Even though these drugs have a small volume of distribution, selective accumulation in renal tubular epithelial cells occurs, so that kidney residues persist in animals for extensive periods. Gentamicin is distributed into synovial fluid in normal horses and local inflammation may increase drug concentrations in the joint and concentrations may increase with repeated doses. Regional perfusion techniques and aminoglycoside‐impregnated polymethyl methacrylate beads are excellent methods of local delivery that avoid the adverse effects of systemic therapy.

Elimination is entirely by renal excretion (glomerular filtration), and unchanged drug is rapidly excreted in the urine. Impaired renal function decreases the rate of excretion and makes it necessary to adjust the dosage interval to prevent accumulation and toxicity. The significant individual variation in pharmacokinetic parameters between animals of the same species exacerbates problems of toxicity with this drug class.

Drug Interactions

Aminoglycosides are commonly additive and sometimes synergistic with beta‐lactam drugs. Synergism does not usually occur in the presence of high‐level plasmid‐mediated or chromosomal resistance. The aminoglycosides are synergistic against streptococci, enterococci, Pseudomonas, and other Gram‐negative bacteria if combined with beta‐lactam antibiotics due to disruption of the bacterial cell wall by the beta‐lactam antibiotic, allowing greater bacterial uptake of the aminoglycoside. Combinations of newer beta‐lactam drugs with newer aminoglycosides provide optimal therapy in seriously ill, neutropenic patients with bacterial infections (Krause et al., 2016).

Aminoglycosides are physically incompatible with a number of drugs including many beta‐lactams, so they should never be mixed in the same syringe. If administered sequentially through an infusion set, care should be taken to flush well between drugs.

Toxicity and Adverse Effects

All aminoglycosides can cause varying degrees of ototoxicity and nephrotoxicity (Table 13.1). Nephrotoxicity (acute tubular necrosis) is the most common adverse effect of aminoglycoside therapy. Neomycin is the most nephrotoxic and streptomycin and dihyrostreptomycin are the least nephrotoxic. Amikacin is often recommended in critical patients over gentamicin as it is considered less nephrotoxic. The aminoglycosides enter the renal tubule after filtration through the glomerulus. From the luminal fluid, the cationic aminoglycoside molecules bind to anionic phospholipids on the proximal tubular cells. The aminoglycoside is taken into the cell via carrier‐mediated pinocytosis and translocated into cytoplasmic vacuoles, which fuse with lysosomes. The drug is sequestered unchanged in the lysosomes. With additional pinocytosis, drug continues to accumulate within the lysosomes. The accumulated aminoglycoside interferes with normal lysosomal function and eventually the overloaded lysosomes swell and rupture. Lysosomal enzymes, phospholipids, and the aminoglycoside are released into the cytosol of the proximal tubular cell, disrupting other organelles and causing cell death (Perazella, 2019) (Figure 13.1).

The risk factors for aminoglycoside toxicity include prolonged therapy (>7–10 days), multiple doses per day, acidosis and electrolyte disturbances (hypokalemia, hyponatremia), volume depletion (shock, endotoxemia), concurrent nephrotoxic drug therapy, age (neonates, geriatrics), preexisting renal disease, and elevated trough concentrations (Mattie et al., 1989).

Calcium supplementation reduces the risk of nephrotoxicity. Nephrotoxicity can also be decreased by feeding a high‐protein/high‐calcium diet such as alfalfa to treated large animals and diets higher than 25% protein to small animals, as protein and calcium cations compete with aminoglycoside cations for binding to renal tubular epithelial cells (Behrend et al., 1994; Schumacher et al., 1991). High dietary protein also increases glomerular filtration rate and renal blood flow, thereby reducing aminoglycoside accumulation.

Because nephrotoxicity is related to aminoglycoside accumulation in the renal proximal tubular cells, it is logical that peak concentrations are not related to toxicity and that longer dosage intervals result in less total drug contact with the renal brush border membrane. High‐dose, once‐daily dosing of aminoglycosides is now common in human and veterinary medicine; it takes advantage of the concentration‐dependent killing and long PAE of these drugs and avoids first‐exposure adaptive resistance and nephrotoxicity.

Serum concentrations of aminoglycosides can be monitored to reduce toxicity and confirm therapeutic concentrations (Bartal et al., 2003). To allow for the distribution phase, blood sampling for the peak concentration is done at 0.5–1 hour after administration and the trough sample is usually taken prior to the next dose. The peak and trough concentrations can then be used to estimate the elimination half‐life for the individual patient. An increase in the elimination half‐life during therapy is a very sensitive indicator of early tubular insult. If using a once‐daily regimen, a blood sample just prior to the next dose may be below the recommended trough concentrations and may even be below the limit of detection of the assay. For these patients, an eight‐hour postdose sample will provide a more accurate estimate of the elimination half‐life. Serum concentrations of drug should be 0.5–2 μg/ml before the next dose (gentamicin, tobramycin) or less than 6 μg/ml for amikacin.

If therapeutic drug monitoring is unavailable, then nephrotoxicity is detected by an increase in urine gamma‐glutamyl transferase (GGT) enzyme and an increase in the urine GGT/urine creatinine (Cr) ratio (van der Harst et al., 2005). The UGGT/UCr may increase to 2–3 times baseline within three days of a nephrotoxic dose. If these tests are not available, the development of proteinuria is the next best indicator of nephrotoxicity and it is easily determined in a practice setting. Elevations in serum urea nitrogen and Cr confirm nephrotoxicity, but are not seen for seven days after significant renal damage has occurred. Elimination half‐lives of 24–45 hours have been reported in horses with renal toxicity, further prolonging the toxic exposure to the drug. While peritoneal dialysis is useful in lowering creatinine and serum urea nitrates, it may not be effective in significantly increasing the elimination of the accumulating aminoglycoside. The animal’s ability to recover most likely depends on the type of medication exposure and the amount of healthy renal tissue remaining to compensate.

Aminoglycoside ototoxicity occurs from the same mechanisms as nephrotoxicity. The tendency to produce vestibular damage (streptomycin, gentamicin) or cochlear damage (amikacin, kanamycin, neomycin) varies with the drug. Tobramycin appears to affect both vestibular (balance) and cochlear (hearing) functions equally. This drug‐specific toxicity may be due to the distribution characteristics of each drug and concentration achieved in each sensory organ. The ototoxic effect of aminoglycosides is potentiated by the loop diuretics furosemide and ethacrynic acid and probably other diuretic agents.

All aminoglycosides given rapidly IV cause bradycardia, reduce cardiac output, and lower blood pressure through an effect on calcium metabolism. These effects are of minor significance (Hague et al., 1997). Neuromuscular blockade is a rare effect, related to blockade of acetylcholine at the nicotinic cholinergic receptor. It is most often seen when anesthetic agents are administered concurrently with aminoglycosides. Affected patients should be treated promptly with parenteral calcium chloride at 10–20 mg/kg IV, calcium gluconate at 30–60 mg/kg IV or neostigmine at 100–200 μg/kg to reverse dyspnea from muscle response depression. Edrophonium at 0.5 mg/kg IV will also reverse neuromuscular blocking effects.

Dosage Considerations

Aminoglycosides produce rapid, concentration‐dependent killing of Gram‐negative aerobes and a prolonged PAE (McKellar et al., 2004). A maximum plasma concentration (Cmax) to MIC ratio is associated with efficacy. A Cmax:MIC ratio of 8–12/1 optimizes bactericidal activity. Higher initial serum concentrations may also be associated with a longer PAE. Traditionally, aminoglycosides were administered every 8–12 hours. If the aminoglycoside is dosed multiple times a day or the drug concentration remains constant, as with a continuous infusion, first‐exposure adaptive resistance persists and increases and the risks of nephrotoxicity and ototoxicity increase. Dose administration at 24‐hour intervals, or longer, may increase efficacy by allowing time for adaptive resistance to reverse. Some clinicians have expressed reservations about once‐daily dosing when intestinal damage allows continued exposure to bacteria that may replicate during the prolonged periods of subtherapeutic aminoglycoside concentrations, but this has not been documented clinically.

Studies in human and veterinary patients support high‐dose, once‐daily therapy of aminoglycosides (Bauquier et al., 2015; Albarellos et al., 2004; Godber et al., 1995; Magdesian et al. 1998; Nestaas et al., 2005). However, the optimal doses and the ideal therapeutic drug monitoring strategy are still unknown (Redpath et al., 2021). All dosage regimens should take into account the patient’s renal function, the exclusive renal excretion route of aminoglycosides, and their toxicity potential. Neonates typically have a higher percentage of extracellular water than adults; therefore, the volume of distribution of aminoglycosides is higher and they typically require higher dosages than adults.

Clinical Use

The toxicity of aminoglycosides has largely restricted their use to the treatment of severe infections. Because there are few alternative treatment options, aminoglycosides are increasingly considered in the treatment of MRSA and MRSP infections in companion animals (Papich, 2012). The more toxic aminoglycosides (e.g., neomycin) are largely restricted to topical or oral use for the treatment of infections caused by Enterobacterales. The less toxic aminoglycosides are usually reserved for the parenteral treatment of severe sepsis caused by Gram‐negative aerobes and, increasingly, the treatment of methicillin‐resistant staphylococcal infections. Of these, gentamicin is usually the first choice followed by amikacin, which due to expense is reserved for sepsis caused by organisms resistant to gentamicin. But even the expensive aminoglycosides can be used for local therapy of musculoskeletal infections. Antimicrobial‐impregnated polymethyl methacrylate beads, collagen sponges, and regional perfusion (intravenous or intraosseous) provide high local concentrations with less expense and reduced risk of systemic toxicity.

Because aminoglycoside residues persist in renal tissues for prolonged periods, the extra‐label use in food animals should be avoided. A voluntary resolution against the extra‐label administration of aminoglycosides has been adopted by the American Association of Bovine Practitioners, the Academy of Veterinary Consultants, the National Cattlemen’s Beef Association and the American Veterinary Medical Association.

Antimicrobial Activity

Streptomycin and dihydrostreptomycin are active against mycobacteria, some mycoplasma, some Gram‐negative rods (including Brucella), and some Staphylococcus aureus. With the exception of mycobacteria, streptomycin is the least active of the aminoglycosides. Among susceptible bacteria are Leptospira, Francisella tularensis, Yersinia pestis, and most Campylobacter fetus ssp. venerealis. Organisms with MIC <4 μg/ml are regarded as susceptible.

Antimicrobial Resistance

Acquired resistance to streptomycin and dihydrostreptomycin is widespread in veterinary pathogens and has virtually eliminated the use of these drugs except for special applications (Alsayeqh et al., 2021). Even arboriculture use of streptomycin selects for multidrug‐resistant nasal and enteric bacterial flora, including extended‐spectrum beta‐lactamase‐producing E. coli (Scherer et al., 2013). Most clinically important resistance is caused by plasmid‐specified enzymes. Through plasmid‐mediated resistance, streptomycin or dihydrostreptomycin resistance genes are commonly linked with sulfonamide, ampicillin, and tetracycline resistance genes. Chromosomal mutations conferring resistance arise commonly in vitro and often in vivo within a few days of treatment, although such mutants may be less viable than their parents.

Drug Interactions

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