β-Lactam Antibiotics

The major structural component of most bacterial cell walls is the peptidoglycan layer. The basic structure is a chain of 10 to 65 disaccharide residues consisting of alternating molecules of N-acetylglucosamine and N-acetylmuramic acid. These chains are then cross-linked with peptide bridges that create a rigid mesh coating for the bacteria. The building of the chains and cross-links is catalyzed by specific enzymes (e.g., transpeptidases, transglycosylases, carboxypeptidases) that are members of a large family of serine proteases. These regulatory enzymes are also called penicillin-binding proteins (PBPs), because they are the targets of β-lactam antibiotics. When growing bacteria are exposed to these antibiotics, the antibiotic binds to specific PBPs in the bacterial cell wall and inhibits assembly of the peptidoglycan chains. This, in turn, activates autolysins that degrade the cell wall, resulting in bacterial cell death. Thus the β-lactam antibiotics generally act as bactericidal agents.

Bacteria can become resistant to β-lactam antibiotics by three general mechanisms: (1) prevention of the interaction between the antibiotic and the target PBP, (2) modification of the binding of the antibiotic to the PBP, and (3) hydrolysis of the antibiotic by bacterial enzymes, β-lactamases. The first mechanism of resistance is seen in gram-negative bacteria (particularly Pseudomonas species). Gram-negative bacteria have an outer membrane that overlies the peptidoglycan layer. Penetration of β-lactam antibiotics into gram-negative rods requires transit through pores in this outer membrane. Changes in the proteins (porins) that form the walls of the pores can alter the size of the pore opening or charge of these channels and result in the exclusion of the antibiotic.

Resistance can also be acquired by modification of the β-lactam antibiotic binding to the PBP. This can be mediated by (1) an overproduction of PBP (a rare occurrence), (2) acquisition of a new PBP (e.g., methicillin resistance in Staphylococcus aureus), or (3) modification of an existing PBP through recombination (e.g., penicillin resistance in Streptococcus pneumoniae) or a point mutation (penicillin resistance in Enterococcus faecium).

Finally, bacteria can produce β-lactamases that inactivate the β-lactam antibiotics. Interestingly, the β-lactamases are in the same family of serine proteases as the PBPs. More than 200 different β-lactamases have been described. Some are specific for penicillins (i.e., penicillinases), cephalosporins (i.e., cephalosporinases), or carbapenems (i.e., carbapenemases), whereas others have a broad range of activity, including some that are capable of inactivating most β-lactam antibiotics. An exhaustive discussion of β-lactamases is beyond the scope of this chapter; however, a brief discussion is germane for understanding the limitations of β-lactam antibiotics. By one classification scheme, β-lactamases have been separated into four classes (A to D). The most common class A β-lactamases are SHV-1 and TEM-1, penicillinases found in common gram-negative rods (e.g., Escherichia, Klebsiella), with minimal activity against cephalosporins. Unfortunately, simple point mutations in the genes encoding these enzymes have created β-lactamases with activity against all penicillins and cephalosporins. These β-lactamases are referred to as extended-spectrum β-lactamases (ESBLs) and are particularly troublesome, because most are encoded on plasmids that can be transferred from organism to organism. The class B β-lactamases are zinc-dependent metalloenzymes that have a broad spectrum of activity against all β-lactam antibiotics, including the cephamycins and carbapenems. The class C β-lactamases are primarily cephalosporinases that are encoded on the bacterial chromosome. Expression of these enzymes is generally repressed, although this can be altered by exposure to certain “inducing” β-lactam antibiotics or by mutations in the genes controlling expression of the enzymes. Expression of this class of β-lactamases is particularly troublesome, because they are active against the most potent expanded-spectrum cephalosporins. The class D β-lactamases are penicillinases found primarily in gram-negative rods.

Glycopeptides

Vancomycin, originally obtained from Streptomyces orientalis, is a complex glycopeptide that disrupts cell wall peptidoglycan synthesis in growing gram-positive bacteria. Vancomycin interacts with the D-alanine-D-alanine termini of the pentapeptide side chains, which interferes sterically with the formation of the bridges between the peptidoglycan chains. Vancomycin is used for the management of infections caused by oxacillin-resistant staphylococci and other gram-positive bacteria resistant to β-lactam antibiotics. Vancomycin is inactive against gram-negative bacteria, because the molecule is too large to pass through the outer membrane pores and reach the peptidoglycan target site. In addition, some organisms are intrinsically resistant to vancomycin (e.g., Leuconostoc, Lactobacillus, Pediococcus, and Erysipelothrix) because the pentapeptide terminates in D-alanine-D-lactate, which does not bind vancomycin. Intrinsic resistance is also found in some species of enterococci that contain a D-alanine-D-serine terminus (i.e., Enterococcus gallinarum, Enterococcus casseliflavus). Finally, some species of enterococci (particularly E. faecium and Enterococcus faecalis) have acquired resistance to vancomycin. The genes for this resistance (primarily vanA and vanB), which also mediate changes in the pentapeptide terminus, can be carried on plasmids and have seriously compromised the usefulness of vancomycin for the treatment of enterococcal infections. More importantly, the gene for vancomycin resistance contained within a transposon on a multiresistance conjugative plasmid has been transferred in vivo from E. faecalis to a multiresistant S. aureus. The transposon then moved from the E. faecalis plasmid and recombined and integrated into the S. aureus resistance plasmid. This resulted in an S. aureus plasmid that encoded resistance to β-lactams, vancomycin, aminoglycosides, and other antibiotics—a plasmid that could be transferred to other staphylococci by conjugation. Interestingly, these resistant strains of Staphylococcus have primarily been restricted to Michigan; however, if this resistance becomes widespread, the medical implications are profound.

Lipopeptides

Daptomycin, a naturally occurring cyclic lipopeptide produced by Streptomyces roseosporus, binds irreversibly to the cytoplasmic membrane, resulting in membrane depolarization and disruption of the ionic gradients, leading to cell death. It has potent activity against gram-positive bacteria, but gram-negative bacteria are resistant to daptomycin, because the drug cannot penetrate through the cell wall to the cytoplasmic membrane. Daptomycin has good activity against multidrug resistant staphylococci, streptococci, and enterococci (including vancomycin-resistant strains.

Polypeptides

Bacitracin, which was isolated from Bacillus licheniformis, is a mixture of polypeptides used in topically applied products (e.g., creams, ointments, sprays) for the treatment of skin infections caused by gram-positive bacteria (particularly those caused by Staphylococcus and group A Streptococcus). Gram-negative bacteria are resistant to this agent. Bacitracin inhibits cell wall synthesis by interfering with dephosphorylation and the recycling of the lipid carrier responsible for moving the peptidoglycan precursors through the cytoplasmic membrane to the cell wall. It may also damage the bacterial cytoplasmic membrane and inhibit ribonucleic acid (RNA) transcription. Resistance to the antibiotic is most likely caused by failure of the antibiotic to penetrate into the bacterial cell.

The polymyxins are a group of cyclic polypeptides derived from Bacillus polymyxa. These antibiotics insert into bacterial membranes like detergents, by interacting with lipopolysaccharides and the phospholipids in the outer membrane, producing increased cell permeability and eventual cell death. Polymyxins B and E (colistin) are capable of causing serious nephrotoxicity. Thus their use has been limited historically to the external treatment of localized infections such as external otitis, eye infections, and skin infections caused by sensitive organisms. However, because some organisms such as Acinetobacter and Pseudomonas are only susceptible to colistin, this antibiotic is used to treat some systemic infections. These antibiotics are most active against gram-negative rods, because gram-positive bacteria do not have an outer membrane.

Isoniazid, Ethionamide, Ethambutol, and Cycloserine

Isoniazid, ethionamide, ethambutol, and cycloserine are cell wall–active antibiotics used for the treatment of mycobacterial infections. Isoniazid (isonicotinic acid hydrazide [INH]) is bactericidal against actively replicating mycobacteria. Although the exact mechanism of action is unknown, the synthesis of mycolic acid is affected (the desaturation of the long-chain fatty acids and the elongation of fatty acids and hydroxy lipids are disrupted). Ethionamide, a derivative of INH, also blocks mycolic acid synthesis. Ethambutol interferes with the synthesis of arabinogalactan in the cell wall, and cycloserine inhibits two enzymes, D-alanine-D-alanine synthetase and alanine racemase, which catalyze cell wall synthesis. Resistance to these four antibiotics results primarily from reduced drug uptake into the bacterial cell or alteration of the target sites.

Aminoglycosides

The aminoglycoside antibiotics (Table 17-5) consist of amino sugars linked through glycosidic bonds to an aminocyclitol ring. Streptomycin, neomycin, kanamycin, and tobramycin were originally isolated from Streptomyces species, and gentamicin and sisomicin were isolated from Micromonospora species. Amikacin and netilmicin are synthetic derivatives of kanamycin and sisomicin, respectively. These antibiotics exert their effort by passing through the bacterial outer membrane (in gram-negative bacteria), cell wall, and cytoplasmic membrane to the cytoplasm, where they inhibit bacterial protein synthesis by irreversibly binding to the 30S ribosomal proteins. This attachment to the ribosomes has two effects: production of aberrant proteins as the result of misreading of the messenger RNA (mRNA), and interruption of protein synthesis by causing the premature release of the ribosome from mRNA.

Table 17-5 Inhibitors of Protein Synthesis

The aminoglycosides are bactericidal because of their ability to bind irreversibly to ribosomes and are commonly used to treat serious infections caused by many gram-negative rods (e.g., Enterobacteriaceae, Pseudomonas, Acinetobacter) and some gram-positive organisms. Penetration through the cytoplasmic membrane is an aerobic, energy-dependent process; so, anaerobes are resistant to aminoglycosides, and susceptible organisms in an anaerobic environment (e.g., abscess) do not respond to treatment. Streptococci and enterococci are resistant to aminoglycosides, because the aminoglycosides fail to penetrate through the cell wall of these bacteria. Treatment of these organisms requires co-administration of an aminoglycoside with an inhibitor of cell wall synthesis (e.g., penicillin, ampicillin, vancomycin) that facilitates uptake of the aminoglycoside.

The most commonly used antibiotics in this class are amikacin, gentamicin, and tobramycin. All three aminoglycosides are used to treat systemic infections caused by susceptible gram-negative bacteria. Amikacin has the best activity and is frequently reserved for treatment of infections caused by gram-negative bacteria that are resistant to gentamicin and tobramycin. Streptomycin is not readily available but has been used for the treatment of tuberculosis, tularemia, and gentamicin-resistant streptococcal or enterococcal infections (in combination with a penicillin).

Resistance to the antibacterial action of aminoglycosides can develop in one of four ways: (1) mutation of the ribosomal binding site, (2) decreased uptake of the antibiotic into the bacterial cell, (3) increased expulsion of the antibiotic from the cell, or (4) enzymatic modification of the antibiotic. The most common mechanism of resistance is enzymatic modification of aminoglycosides. This is accomplished by the action of phosphotransferases (aminoglycoside phosphotransferase [APHs]; seven described), adenyltransferases (adenine nucleotide translocases [ANTs]; four described), and acetyltransferases (acetyl-CoA carboxylases [AACs]; four described) on the amino and hydroxyl groups of the antibiotic. The differences in the antibacterial activity among the aminoglycosides are determined by their relative susceptibility to these enzymes. The other mechanisms by which bacteria develop resistance to aminoglycosides are relatively uncommon. Resistance caused by alteration of the bacterial ribosome requires systematic mutation of the multiple copies of the ribosomal genes that exist in the bacterial cell. Resistance caused by inhibited transport of the antibiotic into the bacterial cell is occasionally observed with Pseudomonas but is more commonly seen with anaerobic bacteria. This mechanism produces low-level cross-resistance to all aminoglycosides. Active efflux of aminoglycosides occurs only in gram-negative bacteria and is rarely observed.

Tetracyclines

The tetracylines (see Table 17-5) are broad-spectrum, bacteriostatic antibiotics that inhibit protein synthesis in bacteria by binding reversibly to the 30S ribosomal subunits, thus blocking the binding of aminoacyl-transfer RNA (tRNA) to the 30S ribosome–mRNA complex. Tetracyclines (i.e., tetracycline, doxycycline, minocycline) are effective in the treatment of infections caused by Chlamydia, Mycoplasma, and Rickettsia species and other selected gram-positive and gram-negative bacteria. All tetracyclines have a similar spectrum of activity, with the primary difference among the antibiotics being in their pharmacokinetic properties (doxycycline and minocycline are easily absorbed and have a long half-life). Resistance to the tetracyclines can stem from decreased penetration of the antibiotic into the bacterial cell, active efflux of the antibiotic out of the cell, alteration of the ribosomal target site, or enzymatic modification of the antibiotic. Mutations in the chromosomal gene encoding the outer membrane porin protein, OmpF, can lead to low-level resistance to the tetracyclines, as well as to other antibiotics (e.g., β-lactams, quinolones, chloramphenicol).

Researchers have identified a variety of genes in different bacteria that control the active efflux of the tetracyclines from the cell. This is the most common cause of resistance. Resistance to the tetracyclines can also result from the production of proteins similar to elongation factors that protect the 30S ribosome. When this happens, the antibiotic can still bind to the ribosome, but protein synthesis is not disrupted.

Glycylclines

Tigecycline, the first representative of this new class of antibiotics, is a semisynthetic derivative of minocycline. It inhibits protein synthesis in the same manner as the tetracyclines. Tigecycline has a higher binding affinity for the ribosome and is less affected by efflux or enzymatic modification. It has a broad spectrum of activity against gram-positive, gram-negative, and anaerobic bacteria, although Proteus, Morganella, Providencia, and P. aeruginosa are generally resistant.

Oxazolidinones

The oxazolidinones are a narrow-spectrum class of antibiotics, with linezolid being the agent currently used. Linezolid blocks initiation of protein synthesis by interfering with the formation of the initiation complex consisting of tRNA, mRNA, and the ribosome. The drug binds to the 50S ribosomal subunit, which distorts the binding site for tRNA, thus inhibiting formation of the 70S initiation complex. Because of this unique mechanism, cross-resistance with other protein inhibitors does not occur. Linezolid has activity against all staphylococci, streptococci, and enterococci (including those strains resistant to penicillins, vancomycin, and the aminoglycosides). Because the multidrug-resistant enterococci are difficult to treat, use of linezolid is generally reserved for these infections.

Chloramphenicol

Chloramphenicol has a broad antibacterial spectrum similar to that of tetracycline but is not commonly used in the United States. The reason for its limited use is that besides interfering with bacterial protein synthesis, it disrupts protein synthesis in human bone marrow cells and can produce blood dyscrasias, such as aplastic anemia (1 per 24,000 treated patients). Chloramphenicol exerts its bacteriostatic effect by binding reversibly to the peptidyl transferase component of the 50S ribosomal subunit, thus blocking peptide elongation. Resistance to chloramphenicol is observed in bacteria producing plasmid-encoded chloramphenicol acetyltransferase, which catalyzes the acetylation of the 3-hydroxy group of chloramphenicol. The product is incapable of binding to the 50S subunit. Less commonly, chromosomal mutations alter the outer membrane porin proteins, causing the gram-negative rods to be less permeable.

Macrolides

Erythromycin, derived from Streptomyces erythreus, is the model macrolide antibiotic (see Table 17-5). The basic structure of this class of antibiotics is a macrocyclic lactone ring bound to two sugars, desosamine and cladinose. Modification of the macrolide structure led to the development of azithromycin, clarithromycin, and roxithromycin. Macrolides exert their effect by their reversible binding to the 23S ribosomal RNA (rRNA) of the 50S ribosomal subunit, which blocks polypeptide elongation. Resistance to macrolides most commonly stems from the methylation of the 23S rRNA, preventing binding by the antibiotic. Other mechanisms of resistance include inactivation of the macrolides by enzymes (e.g., esterases, phosphorylases, glycosidase) or mutations in the 23S rRNA and ribosomal proteins. Macrolides are bacteriostatic antibiotics with a broad spectrum of activity. They have been used to treat pulmonary infections caused by Mycoplasma, Legionella, and Chlamydia species, as well as to treat infections caused by Campylobacter species and gram-positive bacteria in patients allergic to penicillin. Most gram-negative bacteria are resistant to the macrolides. Azithromycin and clarithromycin have also been used to treat infections caused by mycobacteria (e.g., Mycobacterium avium complex).

Ketolides

Ketolides are semisynthetic derivative of erythromycin, modified to increase stability in acid. Telithromycin is currently the only ketolide available for use in the United States. As with the macrolides, telithromycin binds to the 50S ribosomal subunit and blocks protein synthesis. Mutations in 23S rRNA or the ribosomal proteins can lead to resistance. Telithromycin has good activity against staphylococci (except strains that have consititutive resistance to erythromycin), S. pneumoniae, other respiratory pathogens (e.g., H. influenzae, Moraxella catarrhalis), gram-positive rods, and some anaerobes. It is not active against B. fragilis and most aerobic gram-negative rods (e.g., Enterobacteriaceae, Pseudomonas, Acinetobacter, Stenotrophomonas). Telithromycin also has good activity against intracellular pathogens (e.g., Legionella, Mycoplasma, Chlamydia, Chlamydiophila), Rickettsia, Bartonella, Coxiella, Francisella, and M. avium.

Clindamycin

Clindamycin (in the family of lincosamide antibiotics) is a derivative of lincomycin, which was originally isolated from Streptomyces lincolnensis. Like chloramphenicol and the macrolides, clindamycin blocks protein elongation by binding to the 50S ribosome. It inhibits peptidyl transferase by interfering with the binding of the amino acid–acyl-tRNA complex. Clindamycin is active against staphylococci and anaerobic gram-negative rods but is generally inactive against aerobic gram-negative bacteria. Methylation of the 23S rRNA is the source of bacterial resistance. Because both erythromycin and clindamycin can induce this enzymatic resistance (also plasmid mediated), cross-resistance between these two classes of antibiotics is observed.

Streptogramins

The streptogramins are a class of cyclic peptides produced by Streptomyces species. These antibiotics are administered as a combination of two components, group A and group B streptogramins, which act synergistically to inhibit protein synthesis. The antibiotic currently available in this class is quinupristin-dalfopristin. Dalfopristin binds to the 50S ribosomal subunit and induces a conformational change that facilitates binding of quinupristin. Dalfopristin prevents peptide chain elongation, and quinupristin initiates premature release of peptide chains from the ribosome. This combination drug is active against staphylococci, streptococci, and E. faecium (but not E. faecalis). Use of the antibiotic has been restricted primarily to treating vancomycin-resistant E. faecium infections.

Quinolones

The quinolones (Table 17-6) are one of the most widely used classes of antibiotics. These are synthetic chemotherapeutic agents that inhibit bacterial DNA topoisomerase type II (gyrase) or topoisomerase type IV, which are required for DNA replication, recombination, and repair. The DNA gyrase-A subunit is the primary quinolone target in gram-negative bacteria, whereas topoisomerase type IV is the primary target in gram-positive bacteria. The first quinolone used in clinical practice was nalidixic acid. This drug was used to treat urinary tract infections caused by a variety of gram-negative bacteria, but resistance to the drug developed rapidly, causing it to fall out of use. This drug has now been replaced by newer, more active quinolones, such as ciprofloxacin, levofloxacin, and moxifloxacin. Modifying the two-ring quinolone nucleus made these newer quinolones (referred to as fluoroquinolones). These antibiotics have excellent activity against gram-positive and gram-negative bacteria, although resistance can develop rapidly in Pseudomonas, oxacillin-resistant staphylococci, and enterococci. In particular, the newer extended-spectrum quinolones have significant activity against gram-positive bacteria.

Table 17-6 Quinolones

Resistance to the quinolones is mediated by chromosomal mutations in the structural genes for DNA gyrase and topoisomerase type IV. Other mechanisms include decreased drug uptake caused by mutations in the membrane permeability regulatory genes, and overexpression of efflux pumps that actively eliminate the drug. Each of these mechanisms is primarily chromosomally mediated.

Rifampin and Rifabutin

Rifampin, a semisynthetic derivative of rifamycin B produced by Streptomyces mediterranei, binds to DNA-dependent RNA polymerase and inhibits the initiation of RNA synthesis. Rifampin is bactericidal for Mycobacterium tuberculosis and is very active against aerobic gram-positive cocci, including staphylococci and streptococci.

Because resistance can develop rapidly, rifampin is usually combined with one or more other effective antibiotics. Rifampin resistance in gram-positive bacteria results from a mutation in the chromosomal gene that codes for the beta subunit of RNA polymerase. Gram-negative bacteria are resistant intrinsically to rifampin, because of decreased uptake of the hydrophobic antibiotic. Rifabutin, a derivative of rifamycin, has a similar mode and spectrum of activity. It is particularly active against M. avium.

Metronidazole

Metronidazole was originally introduced as an oral agent for the treatment of Trichomonas vaginitis. However, it was also found to be effective in the treatment of amebiasis, giardiasis, and serious anaerobic bacterial infections (including those caused by B. fragilis). Metronidazole has no significant activity against aerobic or facultatively anaerobic bacteria. The antimicrobial properties of metronidazole stem from the reduction of its nitro group by bacterial nitroreductase, thereby producing cytotoxic compounds that disrupt the host DNA. Resistance results either from decreased uptake of the antibiotic or from elimination of the cytotoxic compounds before they can interact with host DNA.

Antimetabolites

The sulfonamides are antimetabolites that compete with p-aminobenzoic acid, thereby preventing the synthesis of the folic acid required by certain microorganisms. Because mammalian organisms do not synthesize folic acid (required as a vitamin), sulfonamides do not interfere with mammalian cell metabolism. Trimethoprim is another antimetabolite that interferes with folic acid metabolism by inhibiting dihydrofolate reductase, thereby preventing the conversion of dihydrofolate to tetrahydrofolate. This inhibition blocks the formation of thymidine, some purines, methionine, and glycine. Trimethoprim is commonly combined with sulfamethoxazole to produce a synergistic combination active at two steps in the synthesis of folic acid. Dapsone and p-aminosalicylic acid are also antifolates that have proved to be useful for treating mycobacterial infections.

Sulfonamides are effective against a broad range of gram-positive and gram-negative organisms, such as Nocardia, Chlamydia, and some protozoa. Short-acting sulfonamides, such as sulfisoxazole, are among the drugs of choice for the treatment of acute urinary tract infections caused by susceptible bacteria, such as E. coli. Trimethoprim-sulfamethoxazole is effective against a large variety of gram-positive and gram-negative microorganisms and is the drug of choice for the treatment of acute and chronic urinary tract infections. The combination is also effective in the treatment of infections caused by Pneumocystis jirovecii, bacterial infections of the lower respiratory tract, otitis media, and uncomplicated gonorrhea.

Resistance to these antibiotics can stem from a variety of mechanisms. Bacteria such as Pseudomonas are resistant as the result of permeability barriers. A decreased affinity of dihydrofolate reductase can be the source of trimethoprim resistance. In addition, bacteria that use exogenous thymidine (e.g., enterococci) are also intrinsically resistant.