Aminoglycosides

Aminoglycosides consist of amino sugars linked through glycosidic bonds to an aminocyclitol. The structures of streptomycin, gentamicin, and other clinically important aminoglycosides are shown in Figure 47-2. The particular amino sugars and specific locations of the amino groups distinguish the compounds and are important for their antimicrobial effects and toxicity. Gentamicin consists of a mixture of three species with little differences in activities.

FIGURE 47–2 Structures of streptomycin and gentamicin and main components of other clinically used aminoglycosides. D, 2-Deoxystreptamine.

The aminoglycosides exert a concentration-dependent bactericidal action by entering the bacterial cell and inhibiting protein synthesis. The overall process consists of two main steps:

The aminoglycosides can cross the complex cell membrane structure of gram-negative bacteria (see Fig. 46-3) and are more effective against aerobic gram-negative than gram-positive bacteria.

Transport of aminoglycosides into bacterial cells involves several steps. These cationic compounds bind to anionic surfaces and penetrate porin channels of the outer membrane of gram-negative bacteria, or the water-filled areas of the peptidoglycan wall in gram-positive bacteria. The aminoglycoside then binds to a molecule in the electron transport chain in the cytoplasmic membrane. The drug-transporter complex is moved across the cytoplasmic membrane by its potential gradient. The transport is an energy-requiring aerobic step that does not occur in an anaerobic environment or at low pH. After crossing the cytoplasmic membrane, the aminoglycosides bind to ribosomes, maintaining a low concentration of intracellular free drug, which facilitates continued drug transfer accumulation. This results in a loss of membrane integrity and eventual death of the bacteria. Ca⁺⁺, Mg⁺⁺, and other divalent ions inhibit the transport of aminoglycosides into bacteria.

Binding to the ribosome leads to inhibition of protein synthesis. This takes place on the ribosomes, where messenger RNA (mRNA) acts as a template for the addition of activated amino acids attached to tRNAs. The 70S ribosomal particles move along the mRNA template, adding the appropriate amino acid (see Fig. 47-1). Aminoglycosides bind to several ribosomal sites (see Table 47-1) of the 30S and 50S subunits of the bacterial ribosome. Streptomycin, the most thoroughly studied, binds to the 30S subunit, although this can be altered by mutation of particular amino acids. Binding of aminoglycosides interferes with protein synthesis in two ways:

Disaggregation of polysomes blocks their ability to move along the mRNA and synthesize a new peptide chain. Aminoglycosides also bind to the juncture between the 30S and 50S subunits and cause distortion of codon recognition, resulting in abnormal protein production. However, the presence of miscoded proteins does not necessarily correlate with cell death, as exemplified by other protein synthesis inhibitors that are bacteriostatic, whereas aminoglycosides are bactericidal. The bactericidal activity of aminoglycosides likely results from a combination of impaired protein synthesis and membrane dysfunction.

Aminoglycosides also demonstrate a prolonged postantibiotic effect, in which suppression of bacterial growth continues after the serum concentration falls below the minimal inhibitory concentration (MIC). Higher aminoglycoside concentrations are associated with a longer postantibiotic effect. This postantibiotic effect has allowed for once-daily dosing of aminoglycosides with a lower risk of associated toxicities.

Synergistic killing has been demonstrated when aminoglycosides are combined with cell wall active agents (e.g., β-lactams, glycopeptides). The explanation for synergy is related partly to increased uptake of the aminoglycosides in the presence of cell wall active agents. Clinically, aminoglycosides and cell wall active agents are combined to achieve synergistic killing against enterococci, Staphylococcus aureus, Pseudomonas aeruginosa, and other Enterobacteriaceae.

Bacterial resistance to aminoglycosides results from:

Clinically, enzymatic modification of the aminoglycosides is the most important mechanism of resistance. Although resistance resulting from altered ribosomes does occur in enterococci, it is rarely seen in gram-negative bacteria and is relatively uncommon. Resistance as a consequence of the inadequate transport of drug across the cytoplasmic membrane is uncommon in aerobic or facultative species but is seen in strict anaerobes. Mutants with alterations in the electron transfer chain and in adenosine triphosphatase activity have been identified, but they are very rare. The resistance of some Pseudomonas species to aminoglycosides may be related to failure of the drug to distort the lipopolysaccharide of the outer membrane, thus not allowing drug to enter the bacterial cell.

The most common form of resistance stems from modification of the aminoglycoside, which occurs through enzyme-catalyzed phosphorylation, adenylation, or acetylation. The genes for these enzymes are located on plasmids or transposons, which can be spread to many different bacterial species. Many such enzymes have been identified, some of which can inactivate only one or two compounds, whereas others can inactivate multiple compounds. For example, an enzyme that acetylates the amino group at position 6 of the amino hexose can inactivate kanamycin, neomycin, tobramycin, amikacin, and netilmicin but not gentamicin or streptomycin. The altered aminoglycosides do not bind as well to ribosomes, and the modified compounds do not trigger accelerated drug uptake.

Aminoglycoside resistance varies by location and local use patterns. A low resistance to gentamicin may occur in one hospital, whereas high resistance may occur in another hospital. It is not feasible at present to predict precise resistance mechanisms. Over prolonged periods, selective use or substitution of one aminoglycoside may lead to reductions in resistance to other aminoglycosides. This was demonstrated at the Minneapolis Veterans Affairs Medical Center, when the repeated selective use of amikacin reduced resistance to gentamicin and tobramycin among aerobic gram-negative organisms. Amikacin is the most resistant of the aminoglycosides to inactivation by resistant organisms, and netilmicin is the second most resistant.

Macrolides, Chloramphenicol, and Clindamycin

The macrolides (erythromycin, clarithromycin, azithromycin, and dirithromycin), chloramphenicol, and clindamycin are discussed as a group because these agents bind to the same site or sites on the ribosomal 50S subunit. They bind to bacterial 70S ribosomes but not to the 80S ribosomes of mammalian cells. Their structures are shown in Figure 47-3. Bacterial resistance is observed for all of these agents.

FIGURE 47–3 Structures of chloramphenicol, erythromycin, and clindamycin.

Erythromycin and the newer macrolides reversibly bind to 50S ribosomal subunits, causing dissociation of peptidyl-tRNA from the ribosome and interference with peptide elongation. Erythromycin inhibits the binding of chloramphenicol to 50S ribosomes, but chloramphenicol does not inhibit erythromycin binding. The activity of macrolides is primarily bacteriostatic; however, bactericidal activity is observed for certain organisms (see Table 47-1).

Bacterial resistance to macrolides occurs by several mechanisms, some of which also confer resistance to clindamycin and streptogramin type B. The most problematic forms of resistance arise either from alteration of ribosomal binding sites or drug efflux. Alteration of ribosomal binding sites occurs via a plasmid-encoded enzyme that methylates the 50S ribosomal subunit. Methylation likely causes a conformational change of the ribosomal target and decreased binding. This type of resistance is associated with the erm (erythromycin ribosome methylation) gene and is referred to as the macrolide-lincosamide-streptogramin B (MLS_B) phenotype, because it confers resistance to macrolides, clindamycin, and streptogramin B. Both erythromycin and clindamycin induce this enzyme, but erythromycin has greater activity. This is clinically important, because an organism resistant to erythromycin and susceptible to clindamycin can become resistant to both drugs during therapy. This applies to the treatment of infections caused by methicillin-resistant Staphylococcus aureus (MRSA), which is often resistant to erythromycin but may appear susceptible to clindamycin. This form of resistance is present on plasmids that can pass from enterococci to streptococci and is encountered in strains of macrolide-resistant Streptococcus pneumoniae and Streptococcus pyogenes.

Efflux systems represent another prominent mechanism of macrolide resistance. This type of resistance is associated with mef genes and does not confer resistance to clindamycin or streptogramin B. For these resistance types, complete cross-resistance exists among erythromycin, clarithromycin, and azithromycin.

Although some gram-negative bacteria possess ribosomes that do not bind erythromycin or clindamycin, the common form of resistance to erythromycin is its failure to pass through the outer membrane of aerobic gram-negative bacteria. For example, 100 times less erythromycin can cross the outer membrane of gram-negative Escherichia coli than that of S. aureus (gram-positive). Gram-negative bacteria may also possess esterases that hydrolyze erythromycin.

Chloramphenicol prevents the addition of new amino acids to growing peptide chains by interfering with binding of the amino acid–acyl–tRNA complex to the 50S subunit, preventing formation of a peptide bond. Chloramphenicol displays primarily bacteriostatic activity but is bactericidal for selected pathogens including Streptococcus pneumoniae, Neisseria meningitidis, and Haemophilus influenzae. Macrolides and clindamycin bind to the same ribosomal site, antagonizing the activity of chloramphenicol, and should not be used concurrently. Most resistance to chloramphenicol is caused by chloramphenicol acetyltransferase. This enzyme catalyzes the acetylation of the hydroxy groups of chloramphenicol, which makes it unable to bind to the 50S subunit. Less common mechanisms of resistance arise from alterations in cell wall permeability or ribosomal proteins.

The mechanism of action of clindamycin is similar to that of erythromycin. The ribosomal binding site for clindamycin overlaps with that of macrolides and chloramphenicol, creating the potential for antagonism when used concurrently. Resistance to clindamycin may arise from alterations in ribosomal binding sites as described, with a resultant MLS_B phenotype conferring resistance to both clindamycin and macrolides. This form of resistance is often plasmid-mediated and has been observed in clindamycin-resistant strains of Bacteroides fragilis. Intrinsic resistance to clindamycin is seen in Enterobacteriaceae and Pseudomonas, resulting from poor permeability of the cell envelope to clindamycin.

Ketolides

The ketolides are semisynthetic derivatives of erythromycin and are part of the MLS_B family of antimicrobials. The currently approved ketolide is telithromycin; its structure is shown in Figure 47-4. Similar to the macrolides, ketolides inhibit protein synthesis at the 50S ribosomal subunit. However, ketolides demonstrate a higher binding affinity. Ketolides are primarily bacteriostatic but demonstrate bactericidal activity against some pathogens, including S. pneumoniae and H. influenzae.

FIGURE 47–4 Structures of telithromycin, quinupristin, dalfopristin, and linezolid.

In contrast to macrolides, ketolides retain activity against most organisms whose ribosomal binding sites have been modified via methylation (MLS_B phenotypes). In addition, ketolides do not appear to induce MLS_B resistance in streptococci but may promote constitutive expression of erm genes in staphylococci with an inducible MLS_B phenotype. Telithromycin-resistant strains of S. pneumoniae have been described in which both ribosomal modification and mutations in ribosomal proteins were present. Telithromycin has retained activity against streptococci demonstrating mef mediated efflux pumps.

Tetracyclines and Glycylcyclines

The structures of the tetracyclines are shown in Figure 47-5. These compounds bind to 30S ribosomes, thereby preventing attachment of the aminoacyl-tRNA to its acceptor site and preventing the addition of amino acids to the peptide chain being synthesized. Differences in the activities of individual tetracyclines are related to their solubility in lipid membranes of the bacteria. These drugs enter the cytoplasm of gram-positive bacteria by an energy-dependent process, but in gram-negative organisms, they pass through the outer membrane by diffusion through porins. Because minocycline and doxycycline are more lipophilic, they can enter gram-negative cells through the outer lipid membrane and through the porins. Once in the periplasmic space, the tetracyclines are transported across the inner cytoplasmic membrane by a protein-carrier system.

FIGURE 47–5 Structures of tetracyclines.

There are several mechanisms of resistance to tetracyclines. The most common, found in both gram-positive and gram-negative bacteria, is plasmid or transposon mediated and involves decreased intracellular accumulation and increased transport of the drug out of the bacterial cell (Fig. 47-6). Drug efflux occurs as a result of the action of a new protein, likely induced by the drug. A second mechanism involves alteration of outer membrane proteins resulting from mutations in chromosomal genes. In a third mechanism the ribosomal binding site is protected as a result of the presence of a plasmid-generated protein that binds to the ribosome. Resistance to one tetracycline usually implies resistance to all tetracyclines. However, some staphylococci and some Bacteroides species are resistant to tetracycline but susceptible to minocycline and doxycycline because of the lipophilicity of these latter agents.

FIGURE 47–6 Mechanism for bacterial resistance to tetracycline (T) caused by efflux.

The glycylcyclines are newly developed tetracyclines that inhibit bacteria previously resistant to all the commercially available tetracyclines. The first of these clinically approved is tigecycline, which binds to the 30S ribosomal subunit with five times greater affinity than the other tetracyclines. Tigecycline is not expelled by the bacterial macrolide or tetracycline pumps. It has been shown to be effective in the treatment of infections caused by MRSA and vancomycin-resistant enterococci (VRE). However, to reduce the risk of resistance developing to tigecycline, its use is restricted to treat infections resistant to the other tetracyclines.

Streptogramins

Streptogramins are a family of compounds derived from Streptomyces pristinaespiralis whose members are classified into two groups based upon structure. For clinical use, two streptogramins, quinupristin

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