Several key steps must be completed for an antimicrobial agent to successfully inhibit or kill an infecting microorganism (Figure 11-1). First, the agent must be in an active form. This is ensured through the pharmacodynamic design of the drug, which takes into account the route by which the patient receives the agent (e.g., orally, intramuscularly, intravenously). Second, the antibiotic must also be able to achieve sufficient levels or concentrations at the site of infection so that it has a chance to exert an antibacterial effect (i.e., it must be in anatomic approximation with the infecting bacteria). The ability to achieve adequate levels depends on the pharmacokinetic properties of the agent, such as rate of absorption, distribution, metabolism, and excretion of the agent’s metabolites. Table 11-1 provides examples of various anatomic limitations characteristic of a few commonly used antibacterial agents. Some agents, such as ampicillin and ceftriaxone, achieve therapeutically effective levels in several body sites, whereas others, such as nitrofurantoin and norfloxacin, are limited to the urinary tract. Therefore, a knowledge of the site of infection can substantially affect the selection of the antimicrobial agent for therapeutic use.

The remaining steps in antimicrobial action relate to direct interactions between the antibacterial agent and the bacterial cell. The antibiotic is attracted to and maintains contact with the cell surface. Because most targets of antibacterial agents are intracellular, uptake of the antibiotic to some location inside the bacterial cell is required. Once the antibiotic has achieved sufficient intracellular concentration, binding to a specific target occurs. This binding involves molecular interactions between the antimicrobial agent and one or more biochemical components that play an important role in the microorganism’s cellular metabolism. Adequate binding of the target results in disruption of cellular processes, leading to cessation of bacterial cell growth and, depending on the antimicrobial agent’s mode of action, cell death. Antimicrobial agents that inhibit bacterial growth but generally do not kill the organism are known as bacteriostatic agents. Effectively reducing the growth rate of an organism provides adequate protection in individuals whose immune system is capable of removing the agent of infection. Agents that usually kill target organisms are said to be bactericidal (Box 11-1). Bacteriocidal agents are more effective against organisms that are more difficult to control in combination with the host’s immune system.

The primary goal in the development and design of antimicrobial agents is to optimize a drug’s ability to efficiently achieve all steps outlined in Figure 11-1 while minimizing toxic effects on human cells and physiology. Different antibacterial agents exhibit substantial specificity in terms of their bacterial cell targets, that is, their mode of action. For this reason, antimicrobial agents are frequently categorized according to their mode of action.

Mode of Action of Antibacterial Agents

The interior of the bacterial cell has several potential antimicrobial targets. However, the processes or structures most frequently targeted are cell wall (peptidoglycan) synthesis, the cell membrane, protein synthesis, metabolic pathways, and DNA and RNA synthesis (Table 11-2).

TABLE 11-2

Summary of Mechanisms of Action for Commonly Used Antibacterial Agents

Antimicrobial Class	Mechanism of Action	Spectrum of Activity
Aminoglycosides (e.g., gentamicin, tobramycin, amikacin, streptomycin, kanamycin)	Inhibit protein synthesis by binding to 30S ribosomal subunit	Gram-positive and gram-negative bacteria; not anaerobic bacteria
β-lactams (e.g., penicillin, ampicillin, mezlocillin, piperacillin, cefazolin, cefotetan, ceftriaxone, cefotaxime, ceftazidime, aztreonam, imipenem)	Inhibit cell wall synthesis by binding enzymes involved in peptidoglycan production (i.e., penicillin-binding proteins [PBPs])	Both gram-positive and gram-negative bacteria, but spectrum may vary with the individual antibiotic.
Chloramphenicol	Inhibits protein synthesis by binding 50S ribosomal subunit	Gram-positive and gram-negative bacteria
Fluoroquinolones (e.g., ciprofloxacin, ofloxacin, norfloxacin)	Inhibit DNA synthesis by binding DNA gyrase and topoisomerase IV	Gram-positive and gram-negative bacteria, but spectrum may vary with individual antibiotic
Glycylglycines (e.g., tigecycline)	Inhibition of protein synthesis by binding to 30S ribosomal subunit	Wide spectrum of gram-positive and gram-negative species including those resistant to tetracycline
Ketolides (e.g., telithromycin)	Inhibition of protein synthesis by binding to 50S ribosomal subunit	Gram-positive cocci including certain macrolide-resistant strains and some fastidious gram-negatives (e.g., H. influenzae and M. catarrhalis)
Lipopeptides (e.g., daptomycin)	Binding and disruption of cell membrane	Gram-positive bacteria including those resistant to beta-lactams and glycopeptides
Nitrofurantoin	Exact mechanism uncertain; may have several bacterial enzyme targets and directly damage DNA	Gram-positive and gram-negative bacteria
Oxazolidinones (e.g., linezolid)	Bind to 50S ribosomal subunit to interfere with initiation of protein synthesis	Wide variety of gram-positive bacteria, including those resistant to other antimicrobial classes
Polymyxins (e.g., polymyxin B and colistin)	Disruption of cell membrane	Gram-negative bacteria, poor activity against most gram-positive bacteria
Rifampin	Inhibits RNA synthesis by binding DNA-dependent, RNA polymerase	Gram-positive and certain gram-negative (e.g., N. meningitidis) bacteria
Streptogramins (e.g., quinupristin/dalfopristin)	Inhibit protein synthesis by binding to two separate sites on the 50S ribosomal subunit	Primarily gram-positive bacteria
Sulfonamides	Interfere with folic acid pathway by binding the enzyme dihydropteroate synthase	Gram-positive and many gram-negative bacteria
Tetracycline	Inhibits protein synthesis by binding 30S ribosomal subunit	Gram-positive and gram-negative bacteria, and several intracellular bacterial pathogens (e.g., chlamydia)
Trimethoprim	Interferes with folic acid pathway by binding the enzyme dihydrofolate reductase	Gram-positive and many gram-negative bacteria

Inhibitors of Cell Wall Synthesis

The bacterial cell wall, also known as the peptidoglycan, or murein, layer, plays an essential role in the life of the bacterial cell. This fact, combined with the lack of a similar structure in human cells, has made the cell wall the focus of attention for the development of bactericidal agents that are relatively nontoxic for humans.

β-Lactam (Beta-Lactam) Antimicrobial Agents.

β-lactam antibiotics have a four-member, nitrogen-containing, β-lactam ring at the core of their structure (Figure 11-2). The antibiotics differ in ring structure and attached chemical groups. This drug class comprises the largest group of antibacterial agents, and dozens of derivatives are available for clinical use. Types of β-lactam agents include penicillins, cephalosporins, carbapenems, and monobactams. The popularity of these agents results from their bactericidal action and lack of toxicity to humans; also, their molecular structures can be manipulated to achieve greater activity for wider therapeutic applications.

The β-lactam ring is the key to the mode of action of these drugs. It is structurally similar to acyl-D-alanyl-D-alanine, the normal substrate required for synthesis of the linear glycopeptide in the bacterial cell wall. The β-lactam binds the enzyme, inhibiting transpeptidation and cell wall synthesis. Most bacterial cells cannot survive once they have lost the capacity to produce and maintain their peptidoglycan layer. The enzymes essential for this function are anchored in the cell membrane and are referred to as penicillin-binding proteins (PBPs). Bacterial species may have four to six different types of PBPs. The PBPs involved in cell wall cross-linking (i.e., transpeptidases) are often the most critical for survival. When β-lactams bind to these PBPs, cell wall synthesis is essentially halted. Death results from osmotic instability caused by faulty cell wall synthesis, or binding of the β-lactam to PBP may trigger a series of events that leads to autolysis and death of the cell.

Because nearly all clinically relevant bacteria have cell walls, β-lactam agents act against a broad spectrum of gram-positive and gram-negative bacteria. However, because of differences among bacteria in their PBP content, natural structural characteristics (e.g., the outer membrane present in gram-negative but not gram-positive bacteria), and their common antimicrobial resistance mechanisms, the effectiveness of β-lactams against different types of bacteria can vary widely. Gram-positive bacteria secrete β-lactamase into the environment, whereas beta-lactamases produced by gram-negative bacteria remain in the periplasmic space, providing increased protection from the antimicrobial. In addition, any given β-lactam drug has a specific group or type of bacteria against which it is considered to have the greatest activity. The type of bacteria against which a particular antimicrobial agent does and does not have activity is referred to as that drug’s spectrum of activity. Many factors contribute to an antibiotic’s spectrum of activity, and knowledge of this spectrum is the key to many aspects of antimicrobial use and laboratory testing.

A common mechanism of bacterial resistance to β-lactams is the production of enzymes (i.e., β-lactamases) that bind and hydrolyze these drugs. Just as there is a variety of β-lactam antibiotics, there is a variety of β-lactamases. The β-lactamases are grouped into four major categories; classes A, B, C, and D. Classes A and D are considered serine peptidases; class C comprises cephalosporinases; and class B, which requires zinc, is called a metallo-β-lactamase. β-lactamase genes should be located on plasmids or transposons, within an integron, or within the chromosome of the organism. An integron is a large cassette region that contains antibiotic resistance genes and the enzyme integrase, which is required for movement of the cassette from one genetic element to another. In addition, the antimicrobial may be constitutively produced, continuously produced, or it may be induced by the presence of a β-lactam.

Bacteria normally susceptible to β-lactams have developed several resistance mechanisms against the antimicrobials. These include genetic mutations in the PBP coding sequence, altering the structure and reducing the binding affinity to the drug; genetic recombination, resulting in a PBP structure resistant to binding of the drug; overproduction of normal PBP, resulting in overload of the drug; and acquiring a new genetic coding sequence for PBP from another organism with a lower affinity to the drug. These acquired types of β-lactam resistance are more commonly found in gram-positive bacteria.

To circumvent the development of antimicrobial resistance, β-lactam combinations comprised of a β-lactam with antimicrobial activity (e.g., ampicillin, amoxicillin, piperacillin) and a beta-lactam without activity capable of binding and inhibiting β-lactamases (e.g., sulbactam, clavulanate, tazobactam) have been developed. The binding β-lactam “ties up” the β-lactamases produced by the bacteria and allows the other β-lactam in the combination to exert its antimicrobial effect. Examples of these β-lactam/β-lactamase inhibitor combinations include ampicillin/sulbactam, amoxicillin/clavulanate, and piperacillin/tazobactam. Such combinations are effective only against organisms that produce β-lactamases that are bound by the inhibitor; they have little effect on resistance that is mediated by altered PBPs (see Mechanisms of Antibiotic Resistance later in this chapter).

Glycopeptides and Lipopeptides.

Glycopeptides are the other major class of antibiotics that inhibit bacterial cell wall synthesis by binding to the end of the peptidoglycan, interfering with transpeptidation. This is a different mechanism from that of the β-lactams, which bind directly to the enzyme. Two such antibiotics, vancomycin and teicoplanin, are large molecules and function differently from β-lactam antibiotics (Figure 11-3). With glycopeptides, the binding interferes with the ability of the PBP enzymes, such as transpeptidases and transglycosylases, to incorporate the precursors into the growing cell wall. With the cessation of cell wall synthesis, cell growth stops and death often follows. Because glycopeptides have a different mode of action, the resistance to β-lactam agents by gram-positive bacteria does not generally hinder their activity. However, because of their relatively large size, they cannot penetrate the outer membrane of most gram-negative bacteria to reach their cell wall precursor targets. Therefore, this agent is usually ineffective against gram-negative bacteria. Teicoplanin is approved for use throughout the world but is not currently available in the United States. When vancomycin is used, its levels should be monitored because the potential for toxicity.

Oritavancin and telavancin, which are lipoglycopeptides, are structurally similar to vancomycin. They are semisynthetic molecules that are glycopeptides that contain hydrophobic chemical groups. However, change in the molecular structure of the lipoglycopeptides provides a mechanism by which they can bind to the bacterial cell membrane, increasing the inhibition of cell wall synthesis. In addition, the lipoglycopeptides increase cell permeability and cause depolarization of the cell membrane potential. These agents also inhibit the transglycosylation process necessary for cell wall synthesis by complexing with the D-alanyl-D-alanine residues. The lipoglycopeptides’ spectrum of activity is comparable to that of vancomycin but also includes vancomycin-intermediate Staphylococcus aureus (VISA).

The lipopeptide daptomycin is the most recently developed antimicrobial capable of exerting its antimicrobial effect by binding and disrupting the cell membrane of gram-positive bacteria. The drug binds to the cytoplasmic membrane and inserts its hydrophobic tail into the membrane, disrupting the permeability and resulting in cell death. Daptomycin has potent activity against gram-positive cocci, including those resistant to other agents such as beta-lactams and glycopeptides (e.g., methicillin-resistant S. aureus [MRSA], vancomycin-resistant enterococci [VRE], and vancomycin-resistant S. aureus [VRSA]). Because of the molecule’s size, daptomycin is unable to penetrate the outer membrane of gram-negative bacilli and thus is ineffective against these organisms.

Several other cell wall–active antibiotics have been discovered and developed over the years, but toxicity to the human host has prevented their widespread clinical use. One example is bacitracin, which inhibits the recycling of certain metabolites required for maintaining peptidoglycan synthesis. Because of potential toxicity, bacitracin is usually only used as a topical antibacterial agent and internal consumption is generally avoided.

Inhibitors of Cell Membrane Function

Polymyxins (polymyxin B and colistin) are cyclic polypeptide agents that disrupt bacterial cell membranes. The polymyxins act as detergents, interacting with the phospholipids in the cell membranes and increasing permeability. This disruption results in leakage of macromolecules and ions essential for cell survival. Because their effectiveness varies with the molecular makeup of the bacterial cell membrane, polymyxins are not equally effective against all bacteria. Most notably, they are more effective against gram-negative bacteria, whereas activity against gram-positive bacteria tends to be poor. Furthermore, human host cells also have membranes, therefore polymyxins pose a risk of toxicity. The major side effects are neurotoxicity and nephrotoxicity. Although toxic, the polymyxins are often the antimicrobial agents of last resort when gram-negative bacilli (e.g., Pseudomonas aeruginosa, Acinetobacter spp.) that are resistant to all other available agents are encountered.

Inhibitors of Protein Synthesis

Several classes of antibiotics target bacterial protein synthesis and severely disrupt cellular metabolism. Antibiotic classes that act by inhibiting protein synthesis include aminoglycosides, macrolide-lincosamide-streptogramins (MLS group), ketolides (e.g., telithromycin) chloramphenicol, tetracyclines, glycylglycines (e.g., tigecycline), and oxazolidinones (e.g., linezolid). Although these antibiotics are generally categorized as protein synthesis inhibitors, the specific mechanisms by which they inhibit protein synthesis differ significantly.

Aminoglycosides and Aminocyclitols.

Aminoglycosides (aminoglycosidic aminocyclitol) inhibit bacterial protein synthesis by irreversibly binding to protein receptors on the organism’s 30S ribosomal subunit. This process interrupts several steps, including initial formation of the protein synthesis complex, accurate reading of the messenger RNA (mRNA) code, and formation of the ribosomal-mRNA complex. The structure of a commonly used aminoglycoside, gentamicin, is shown in Figure 11-4. Other aminoglycosides include tobramycin, amikacin, streptomycin, and kanamycin. The spectrum of activity of aminoglycosides includes a wide variety of aerobic gram-negative and certain gram-positive bacteria, such as S. aureus. Bacterial uptake of the aminoglycosides is accomplished by using them in combination with cell wall–active antibiotics, such as β-lactams or vancomycin. Anaerobic bacteria are unable to uptake these agents intracellularly and therefore are typically not inhibited by aminoglycosides. Aminoglycosides are associated with toxicity, and blood levels should be monitored during therapy. The major toxicities are nephrotoxicity and auditory or vestibular toxicity.

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