Folic Acid Synthesis and Regeneration

The bacterial synthesis of folic acid involves a multistep enzyme-catalyzed reaction sequence (Fig. 48-1). Tetrahydrofolic acid is the physiologically active form of folic acid and is required as a cofactor in synthesis of thymidine, purines, and bacterial DNA. Sulfonamides are structural analogs of p-aminobenzoic acid and competitively inhibit dihydropteroate synthase. TMP blocks the production of tetrahydrofolate from dihydrofolate by reversibly inhibiting the required enzyme, dihydrofolate reductase. Thus these two drugs block the synthesis of tetrahydrofolate at different steps in the synthetic pathway and result in a bactericidal synergistic effect.

FIGURE 48–1 Folate synthesis pathway and sites of action of trimethoprim and sulfamethoxazole.

From Masters PA, et al. Trimethoprim-sulfamethoxazole revisited. Arch Intern Med 2003; 163:402. Copyright 2003 American Medical Association. All rights reserved.

Sulfonamides

The sulfonamides are bacteriostatic because microorganisms must synthesize their own folic acid, whereas mammalian cells do not. When folate synthesis is inhibited, bacterial cell growth is halted. This inhibition can be reversed by addition of purines, thymidine, methionine, and serine. Resistance to sulfonamides is widespread, and its incidence continues to increase among all major bacterial pathogens. One mechanism is reduced cellular uptake of the drug, which can be chromosomal or plasmid in origin. A second mechanism is altered dihydropteroate synthetase, which can result from a point mutation or the presence of a plasmid that causes synthesis of a new enzyme. Replacement of a single amino acid in the enzyme alters its affinity for sulfonamides. In enteric species, plasmid-propagated resistance is the common form. A final mechanism of resistance is the production of increased amounts of p-aminobenzoic acid. This mechanism is exhibited by some staphylococci but is not common. Resistance stemming from an altered enzyme can develop during therapy.

Trimethoprim

TMP was used initially as an antimalarial drug but has been replaced by pyrimethamine, which acts by a similar mechanism. The antimalarial and antibacterial actions of TMP stem from its high affinity for bacterial dihydrofolate reductase. TMP binds competitively and inhibits this enzyme in bacterial and mammalian cells. Approximately 100,000 times higher concentrations of drug are needed to inhibit the human enzyme as compared with the bacterial enzyme. This enzyme is also inhibited by methotrexate, discussed in Chapter 54. TMP thus prevents conversion of dihydrofolate to tetrahydrofolate and blocks formation of thymidine, some purines, methionine, and glycine in bacteria, leading to rapid death of the microorganisms.

TMP and SMX are used effectively in combination to achieve synergistic effects, which they accomplish by blocking different steps in folic acid synthesis. Moreover, sulfamethoxazole potentiates the action of TMP by reducing the dihydrofolate competing with TMP for binding to dihydrofolate reductase. The combination of the two drugs is bactericidal.

Resistance to TMP and to the combination of TMP-SMX stems from permeability changes and from the presence of an altered dihydrofolate reductase. Production of this enzyme can be modified by a chromosomal mutation or by a plasmid. There is an increasing incidence of resistance to TMP-SMX by the plasmid mechanism. A mutation to thymine dependence has also been found, as has an overproduction of dihydrofolate reductase.

Fluoroquinolones

The fluoroquinolones include norfloxacin, ciprofloxacin, levofloxacin, moxifloxacin, and ofloxacin. Fluoroquinolones all have a fluorine at position 6 in the 2 ring structure (Fig. 48-2). Gatifloxacin was recently withdrawn from the market in the United States.

FIGURE 48–2 Basic 2-ring structure of fluoroquinolones. All fluoroquinolones have a fluorine at position 6 in the 2-ring structure. Other substitutions at positions 1 to 5 and 7 to 8 are associated with changes in antibacterial spectrum and pharmacokinetics.

The fluoroquinolones act by inhibiting type 2 bacterial DNA topoisomerases, DNA gyrase, and topoisomerase IV. These topoisomerases are enzymes that consist of α- and β-subunits (encoded for by gyrA and gyrB or parC and parE, respectively) and catalyze the direction and extent of supercoiling and other topological reactions of DNA chains. Fluoroquinolones act by binding to and trapping the enzyme-DNA complex. This trapped complex blocks DNA synthesis and cell growth and ultimately has a lethal effect on the cell, possibly by releasing lethal double-strand DNA breaks from the complex. The primary target for the quinolones is determined by the differing sensitivities of DNA gyrase and topoisomerase IV to the particular quinolone in each organism (Fig. 48-3).

FIGURE 48–3 Mechanism of cytotoxicity by quinolones. Topoisomerases bind to DNA in a noncovalent fashion followed by formation of transient cleavage complexes. In these complexes, type 2 topoisomerase (DNA gyrase or topoisomerase IV) creates double-stranded breaks. In the presence of quinolones, levels of cleavage complexes (shown in brackets) increase dramatically. After traversal by replication complexes or helicases, transient topoisomerase-mediated breaks become permanent double-stranded fractures, triggering events that ultimately culminate in cell death.

Data from Froelich-Ammon SJ, Osheroff N. Increased drug affinity as the mechanistic basis for drug hypersensitivity of a mutant type II topoisomerase. J Biological Chem 1995; 270[47]:28018-28021.

Bacterial resistance is the most common and serious problem confronting the clinical use of fluoroquinolones. Mutations in the type 2 topoisomerases DNA gyrase or topoisomerase IV account for most bacterial resistance to fluoroquinolones. Stepwise increases in resistance are associated with sequential mutations in gyrA (or gyrB) and parC (or parE). Decreased permeability, active efflux, and plasmid-mediated resistance have also been described. Fluoroquinolone resistance of clinical significance occurs in Staphylococcus aureus, Pseudomonas aeruginosa, Campylobacter spp., E. coli and other Enterobacteriaceae, N. gonorrhea and, more recently, Streptococcus pneumoniae. Higher rates of fluoroquinolone resistance in a population are often associated with high rates of fluoroquinolone use, implicating selection of spontaneous mutants. However, community spread of single clones of fluoroquinolone-resistant S. pneumoniae has recently been observed.

Nitrofurans

Nitrofurantoin is a member of a group of synthetic nitrofuran compounds that also includes nitrofurazone. The precise mechanism of action of the nitrofurans is not established. They inhibit many bacterial enzyme systems, most probably through DNA damage. A nitroreductase bacterial enzyme converts the compounds to short-lived intermediates, including oxygen free radicals, which interact with DNA to cause strand breakage and bacterial damage.

Resistance develops infrequently. It is not plasmid-mediated, but appears to result from a mutation associated with a loss of bacterial nitroreductase activity.

Polymyxins

Polymyxins are detergents with both lipophilic and lipophobic groups that interact with phospholipids and disrupt bacterial cell membranes. The initial damage is to the cell wall, with a subsequent loss of periplasmic enzymes. The divalent cationic sites on the lipopolysaccharide component of the outer membrane of gram-negative organisms interact with the amino groups of the cyclic polymyxin peptide. The fatty acid tail portion of the drug molecule penetrates into the hydrophobic areas of the outer wall to produce holes in the membrane, through which intracellular constituents leak out of the bacteria (Fig. 48-4). A bacterium is rendered susceptible to the agent as a result of phospholipids in the bacteria cell wall interacting with the drug. The cell walls of resistant bacteria restrict the transport of polymyxin and prevent access of the drug to the cell membrane. Elevated concentrations of Ca⁺⁺ or Mg⁺⁺ reduce the activity of the polymyxins.

FIGURE 48–4 Mechanism of action of polymyxins. Microbial cell in absence (A) and presence (B) of polymyxin.

Sulfonamides

Sulfonamides are generally well absorbed from the gastrointestinal (GI) tract, with most absorption occurring in the small intestine. There is minimal absorption from topical application.

Sulfonamides differ in their protein-binding capacity, from a low of 45% for sulfadiazine to >90% for sulfisoxazole, sulfasalazine, and sulfadoxine, with less protein binding in renal failure. The drugs enter most body compartments, including ocular, pleural, peritoneal, synovial, and cerebrospinal fluid (CSF). Highest concentrations in the CSF are achieved with sulfadiazine, reaching 30% to 80% of plasma concentrations. Sulfonamides cross the placenta and enter the fetal circulation.

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