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.

TABLE 11-1

Anatomic Distribution of Some Common Antibacterial Agents

	Serum-Blood*	Cerebrospinal Fluid	Urine
Ampicillin	+	+	+
Ceftriaxone	+	+	+
Vancomycin	+	±	+
Ciprofloxacin	+	±	+
Gentamicin	+	−	+
Clindamycin	+	−	−
Norfloxacin	−	−	+
Nitrofurantoin	−	−	+

+, Therapeutic levels generally achievable at that site; ±, therapeutic achievable levels moderate to poor; −, therapeutic levels generally not achievable at that site.

^*Serum-blood represents a general anatomic distribution.

Figure 11-1 The basic steps required for antimicrobial activity and strategic points for bacterial circumvention or interference (marked by X) of antimicrobial action, leading to resistance.

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.

Figure 11-2 Basic structures and examples of commonly used β-lactam antibiotics. The core β-lactam ring is highlighted in yellow in each structure. (Modified from Salyers AA, Whitt DD, editors: *Bacterial pathogenesis: a molecular approach,* Washington, DC, 1994, ASM Press.)

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.

Figure 11-3 Structure of vancomycin, a non–β-lactam antibiotic that inhibits cell wall synthesis. (Modified from Salyers AA, Whitt DD, editors: *Bacterial pathogenesis: a molecular approach,* Washington, DC, 1994, ASM Press.)

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.

Figure 11-4 Structure of the commonly used aminoglycoside gentamicin. Potential sites of modification by adenylating, phosphorylating, and acetylating enzymes produced by bacteria are highlighted. (Modified from Salyers AA, Whitt DD, editors: *Bacterial pathogenesis: a molecular approach,* Washington, DC, 1994, ASM Press.)

Fluoroquinolones.

Fluoroquinolones, also often simply referred to as quinolones, are derivatives of nalidixic acid, an older antibacterial agent. The structures of two quinolones, ciprofloxacin and ofloxacin, are shown in Figure 11-5. These agents bind to and interfere with DNA gyrase enzymes involved in the regulation of bacterial DNA supercoiling, a process essential for DNA replication, recombination, and repair. The newer fluoroquinolones also inhibit topoisomerase IV. Topoisomerase IV functions very similarly to DNA gyrase, unlinking DNA after replication. The fluoroquinolones are potent bactericidal agents and have a broad spectrum of activity that includes gram-negative and gram-positive organisms. The fluoroquinolones target the DNA gyrase in gram-negative organisms and topoisomerase IV in gram-positive organisms. Because these agents interfere with DNA replication and therefore cell division, the drugs are bacteriocidal. However, the spectrum of activity varies with the individual quinolone agent. Toxicity varies with a variety of factors. Tendinitis and rupture of the Achilles tendon have been associated with fluoroquinolone treatment in the general population, and the risk is greater in older patients.

Metronidazole.

The exact mechanism of metronidazole’s antibacterial activity is related to the presence of a nitro group in the chemical structure. The nitro group is reduced by a nitroreductase in the bacterial cytoplasm, generating cytotoxic compounds and free radicals that disrupt the host DNA. Activation of metronidazole requires reduction under conditions of low redox potential, such as are found in anaerobic environments. Therefore, this agent is most potent against anaerobic and microaerophilic organisms, notably those that are gram negative. The drug is also effective in the treatment of protozoans, including Trichomonas and Giardia spp. and Entamoeba histolytica. Because susceptibility testing is not routinely performed on anaerobes, resistance is underreported. An emerging resistance to metronidazole is creating difficulties associated with bacterial diagnostics and treatments. Toxicity is low. Adverse side effects generally include mild gastrointestinal symptoms.

Rifamycin.

Rifamycins, which include the drug rifampin (also known as rifampicin), are semisynthetic antibiotics that bind to the enzyme DNA-dependent RNA polymerase and inhibit synthesis of RNA. Because rifampin does not effectively penetrate the outer membrane of all gram-negative bacteria, activity against these organisms is decreased compared to the drug’s activity in gram-positive bacteria. In addition, spontaneous mutation, resulting in the production of rifampin-insensitive RNA polymerases, occurs at a relatively high frequency of mutation. Therefore, rifampin is typically used in combination with other antimicrobial agents. Rifampin’s side effects include gastrointestinal symptoms and hypersensitivity reactions.

Inhibitors of Other Metabolic Processes

Antimicrobial agents that target bacterial processes other than those already discussed include sulfonamides, trimethoprim, and nitrofurantoin.

Sulfonamides.

The bacterial folic acid pathway produces precursors required for DNA synthesis (Figure 11-6). Sulfonamides target and bind to one of the enzymes, dihydropteroate synthase, and disrupt the folic acid pathway. Several different sulfonamide derivatives are available for clinical use. These agents are active against a wide variety of bacteria, including the gram-positive and gram-negative (except P. aeruginosa) species. Sulfonamides are moderately toxic, causing vomiting, nausea, and hypersensitivity reactions. Sulfonamides are also antagonistic for several other medications, including warfarin, phenytoin, and oral hypoglycemic agents.

Trimethoprim.

Like the sulfonamides, trimethoprim targets the folic acid pathway. However, it inhibits a different enzyme, dihydrofolate reductase (see Figure 11-6). Trimethoprim is active against several gram-positive and gram-negative species. Frequently, trimethoprim is combined with a sulfonamide (usually sulfamethoxazole) into a single formulation to produce an antibacterial agent that can simultaneously attack two targets on the same folic acid metabolic pathway. This drug combination can enhance activity against various bacteria and may help prevent the emergence of bacterial resistance to a single agent. Toxicity is typically mild. Adverse side effects include gastrointestinal symptoms and allergic skin rashes. Patients with acquired immunodeficiency syndrome (AIDS) develop side effects more often than healthy individuals.

Nitrofurantoin.

Nitrofurantoin consists of a nitro group on a heterocyclic ring. The mechanism of action of nitrofurantoin is diverse and multifaceted. This agent may have several targets involved in bacterial protein and enzyme synthesis. Nitrofurantoin is converted by bacterial nitroreductases to reactive intermediates that bind bacterial ribosomal proteins and rRNA, disrupting synthesis of RNA, DNA, and proteins. Nitrofurantoin is used to treat uncomplicated urinary tract infections and has good activity against most of the gram-positive and gram-negative bacteria that cause infections at that site. Toxicity primarily consists of gastrointestinal symptoms, including diarrhea, nausea, and vomiting. Chronic pulmonary conditions may develop, including irreversible pulmonary fibrosis.

Mechanisms of Antibiotic Resistance

Principles

Successful bacterial resistance to antimicrobial action requires interruption or disturbance of one or more steps essential for effective antimicrobial action (see Figure 11-1). These disturbances or resistance mechanisms can occur as a result of various processes, but the end result is partial or complete loss of antibiotic effectiveness. Different aspects of antimicrobial resistance mechanisms discussed include biologic versus clinical antimicrobial resistance, environmentally mediated antimicrobial resistance, and microorganism-mediated antimicrobial resistance.

Biologic Versus Clinical Resistance

The development of bacterial resistance to antimicrobial agents to which they were originally susceptible requires alterations in the cell’s physiology or structure. Biologic resistance refers to changes that result in observably reduced susceptibility of an organism to a particular antimicrobial agent. When antimicrobial susceptibility has been lost to such an extent that the drug is no longer effective for clinical use, the organism has achieved clinical resistance.

It is important to note that biologic resistance and clinical resistance do not necessarily coincide. In fact, because most laboratory methods used to detect resistance focus on detecting clinical resistance, microorganisms may undergo substantial change in their levels of biologic resistance without notice. For example, for some time Streptococcus pneumoniae, a common cause of pneumonia and meningitis, was inhibited by penicillin at concentrations of 0.03 µg/mL or less. The clinical laboratory focused on the ability to detect strains requiring 2 µg/mL of penicillin or more for inhibition; this was the defined threshold for resistance required for interference with effective treatment using penicillin. However, although no isolates were being detected that required more than 2 µg/mL of penicillin for inhibition, strains were developing biologic resistance that required penicillin concentrations 10 to 50 times higher than 0.03 µg/mL for inhibition.

From a clinical laboratory and public health perspective, it is important to realize that biologic development of antimicrobial resistance is an ongoing process. Our inability to reliably detect all these processes with current laboratory procedures and criteria should not be misinterpreted as evidence that no changes in biologic resistance are occurring.

Environmentally Mediated Antimicrobial Resistance

Antimicrobial resistance is the result of nearly inseparable interactions involving the drug, the microorganism, and the environment in which they coexist. Characteristics of the antimicrobial agents, other than the mode and spectrum of activity, include important aspects of each drug’s pharmacologic attributes. However these factors are beyond the scope of this text. Microorganism characteristics are discussed in subsequent sections of this chapter (see Microorganism-Mediated Antimicrobial Resistance). The environmental impact on antimicrobial activity is considered here, and its importance cannot be overstated.

Environmentally mediated resistance is defined as resistance directly resulting from physical or chemical characteristics of the environment that either directly alter the antimicrobial agent or alter the microorganism’s normal physiologic response to the drug. Examples of environmental factors that mediate resistance include pH, anaerobic atmosphere, cation concentrations, and thymidine content.

Several antibiotics are affected by the pH of the environment. For instance, the antibacterial activities of erythromycin and aminoglycosides diminish with decreasing pH, whereas the activity of tetracycline decreases with increasing pH.

Aminoglycoside-mediated shutdown of bacterial protein synthesis requires intracellular uptake across the cell membrane. Most of the aminoglycoside uptake is driven through oxidative processes in the cell. In the absence of oxygen, uptake (and hence the activity of the aminoglycoside) is substantially diminished.

Aminoglycoside activity is also affected by the concentration of cations in the environment, such as calcium and magnesium (Ca++ and Mg++). This effect is most notable with P. aeruginosa. As shown in Figure 11-1, an important step in antimicrobial activity is the adsorption of the antibiotic to the bacterial cell surface. Aminoglycoside molecules have a net positive charge, and as is true for most gram-negative bacteria, the outer membrane of P. aeruginosa has a net negative charge. This electrostatic attraction facilitates attachment of the drug to the surface before internalization and subsequent inhibition of protein synthesis (Figure 11-7). However, calcium and magnesium cations compete with the aminoglycosides for negatively charged binding sites on the cell surface. If the positively charged calcium and magnesium ions outcompete aminoglycoside molecules for these sites, the amount of the drug taken up is decreased and antimicrobial activity is diminished. For this reason, aminoglycoside activity against P. aeruginosa tends to decrease as environmental cation concentrations increase.

Figure 11-7 Cations (Mg++ and Ca++) and aminoglycosides (AG++) compete for the negatively charged binding sites on the outer membrane surface of *Pseudomonas aeruginosa.* Such competition is an example of the impact that environmental factors (e.g., cation concentrations) can have on the antibacterial activity of aminoglycosides.

The presence of certain metabolites or nutrients in the environment may also affect antimicrobial activity. For example, enterococci are able to use thymine and other exogenous folic acid metabolites to circumvent the activities of the sulfonamides and trimethoprim, which are folic acid pathway inhibitors (see Figure 11-6). In essence, if the environment supplies other metabolites for the microorganism, the activities of antibiotics that target pathways for producing those metabolites are greatly diminished, if not entirely lost. In the absence of the metabolites, full susceptibility to the antibiotics may be restored.

Information about environmentally mediated resistance is used to establish standardized testing methods that minimize the impact of environmental factors, allowing more accurate determination of microorganism-mediated resistance mechanisms (see the following discussion). It is important to note that in vitro, testing conditions are not established to recreate the in vivo physiology of infection, but rather are set to optimize detection of resistance expressed by microorganisms.

Microorganism-Mediated Antimicrobial Resistance

Microorganism-mediated resistance refers to antimicrobial resistance that results from genetically encoded traits of the microorganism. Organism-based resistance can be divided into two subcategories, intrinsic or inherent resistance and acquired resistance.

Intrinsic Resistance

Antimicrobial resistance resulting from the normal genetic, structural, or physiologic state of a microorganism is referred to as intrinsic resistance (Table 11-3). Such resistance is considered a natural and consistently inherited characteristic associated with the vast majority of strains in a particular bacterial group, genus, or species. Therefore, this resistance pattern may be predictable, leading to identification of the organism. Intrinsic resistance profiles are useful for determining which antimicrobial agents should be included in the battery of drugs tested against specific types of organisms. For example, referring to the information given in Table 11-3, aztreonam would not be included in antibiotic batteries tested against gram-positive cocci. Similarly, vancomycin would not be routinely tested against gram-negative bacilli. As is discussed in Chapter 7, intrinsic resistance profiles are also useful markers to aid the identification of certain bacteria or bacterial groups.

TABLE 11-3

Examples of Intrinsic Resistance to Antibacterial Agents

Natural Resistance	Mechanism
Anaerobic bacteria versus aminoglycosides	Lack of oxidative metabolism to drive uptake of aminoglycosides
Gram-positive bacteria versus aztreonam (β-lactam)	Lack of penicillin-binding proteins (PBPs) that bind and are inhibited by this β-lactam antibiotic
Gram-negative bacteria versus vancomycin	Lack of uptake resulting from inability of vancomycin to penetrate outer membrane
Pseudomonas aeruginosa versus sulfonamides, trimethoprim, tetracycline, or chloramphenicol	Lack of uptake resulting from inability of antibiotics to achieve effective intracellular concentrations
Klebsiella spp. versus ampicillin (a β-lactam) targets	Production of enzymes (β-lactamases) that destroy ampicillin before the drug can reach the PBP
Aerobic bacteria versus metronidazole	Inability to anaerobically reduce drug to its active form
Enterococci versus aminoglycosides	Lack of sufficient oxidative metabolism to drive uptake of aminoglycosides
Enterococci versus all cephalosporin antibiotics	Lack of PBPs that effectively bind and are inhibited by these lactams
Lactobacilli and Leuconostoc sp. versus vancomycin	Lack of appropriate cell wall precursor target to allow vancomycin to bind and inhibit cell wall synthesis
Stenotrophomonas maltophilia versus imipenem (a beta-lactam)	Production of enzymes (β-lactamases) that destroy imipenem before the drug can reach the PBP targets

Acquired Resistance

Antibiotic resistance resulting from altered cellular physiology and structure caused by changes in a microorganism’s genetic makeup is known as acquired resistance. Unlike intrinsic resistance, acquired resistance may be a trait associated with specific strains of a particular organism group or species. Therefore, the presence of this type of resistance in any clinical isolate is unpredictable. This unpredictability is the primary reason laboratory methods are necessary to detect resistance patterns in clinical isolates.

Because acquired resistance mechanisms are all genetically encoded, the methods for acquisition involve genetic change or exchange. Therefore, resistance may be acquired by:

• Successful genetic mutation

• Acquisition of genes from other organisms via gene transfer mechanisms

• A combination of mutational and gene transfer events

Common Pathways for Antimicrobial Resistance

Whether resistance is intrinsic or acquired, bacteria share similar pathways or strategies to effect resistance to antimicrobial agents. Of the pathways listed in Figure 11-8, those that involve enzymatic destruction or alteration of the antibiotic, decreased intracellular uptake or accumulation of drug, and altered antibiotic target are the most common. One or more of these pathways may be expressed by a single cell successfully avoiding and protecting itself from the action of one or more antibiotics.

Figure 11-8 Overview of common pathways bacteria use to effect antimicrobial resistance.

Resistance to Beta-Lactam Antibiotics

As discussed earlier, bacterial resistance to beta-lactams may be mediated by enzymatic destruction of the antibiotics (β-lactamase); altered antibiotic targets, resulting in low affinity or decreased binding of antibiotic to the target PBPs; or decreased intracellular uptake or increased cellular efflux of the drug (Table 11-4). All three pathways play an important role in clinically relevant antibacterial resistance, but bacterial destruction of β-lactams through the production of β-lactamases is by far the most common method of resistance. Extended spectrum β-lactamases are derived from β-lactamases and confer resistance to both penicillins and cephalosporins; carbapenemases are active against carbapenem drugs, such as imipenem. β-lactamases open the drug’s β-lactam ring, and the altered structure prevents subsequent effective binding to PBPs; consequently, cell wall synthesis is able to continue (Figure 11-9).

TABLE 11-4

Summary of Resistance Mechanisms for Beta-Lactams, Vancomycin, Aminoglycosides, and Fluoroquinolones

Antimicrobial Class	Resistance Pathway	Specific Mechanism	Examples
β-lactams (e.g., penicillin, ampicillin, mezlocillin, piperacillin, cefazolin, cefotetan, ceftriaxone, cefotaxime, ceftazidime, aztreonam, imipenem)	Enzymatic destruction	β-lactamase enzymes destroy β-lactam ring, thus antibiotic cannot bind to penicillin-binding protein (PBP) and interfere with cell wall synthesis (see Figure 11-9)	Staphylococcal resistance to penicillin; resistance of Enterobacteriaceae and Pseudomonas aeruginosa to several penicillins, cephalosporins, and aztreonam
	Altered target	Mutational changes in original PBPs or acquisition of different PBPs that do not bind β-lactams sufficiently to inhibit cell wall synthesis	Staphylococcal resistance to methicillin and other available β-lactams Penicillin and cephalosporin resistance in Streptococcus pneumoniae and viridans streptococci
	Decreased uptake	Porin channels (through which β-lactams cross the outer membrane to reach PBPs of gram-negative bacteria) change in number or character so that β-lactam uptake is substantially diminished	P. aeruginosa resistance to imipenem
Glycopeptides (e.g., vancomycin)	Altered target	Alteration in the molecular structure of cell wall precursor components decreases binding of vancomycin so that cell wall synthesis is able to continue	Enterococcal and Staphylococcus aureus resistance to vancomycin
Glycopeptides (e.g., vancomycin)	Target overproduction	Excess peptidoglycan	Vancomycin-intermediate staphylococci
Aminoglycosides (e.g., gentamicin, tobramycin, amikacin, streptomycin, kanamycin)	Enzymatic modification	Modifying enzymes alter various sites on the aminoglycoside molecule so that the ability of drug to bind the ribosome and halt protein synthesis is greatly diminished or lost	Gram-positive and gram-negative resistance to aminoglycosides
	Decreased uptake	Porin channels (through which aminoglycosides cross the outer membrane to reach the ribosomes of gram-negative bacteria) change in number or character so that aminoglycoside uptake is substantially diminished	Aminoglycoside resistance in a variety of gram-negative bacteria
	Altered target	Mutational changes in ribosomal binding site diminish ability of aminoglycoside to bind sufficiently and halt protein synthesis	Enterococcal resistance to streptomycin (may also be mediated by enzymatic modifications)
Quinolones (e.g., ciprofloxacin, ofloxacin, levofloxacin, norfloxacin, lomefloxacin)	Decreased uptake	Alterations in the outer membrane diminish uptake of drug and/or activation of an “efflux” pump that removes quinolones before an intracellular concentration sufficient to inhibit DNA metabolism can be achieved	Gram-negative and staphylococcal (efflux mechanism only) resistance to various quinolones
	Altered target	Changes in the DNA gyrase subunits decrease ability of quinolones to bind this enzyme and interfere with DNA processes	Gram-negative and gram-positive resistance to various quinolones
Macrolides (e.g., erythromycin, azithromycin, clarithromycin)	Efflux	Pumps drug out of cell before target binding	Various streptococci and staphylococci
	Altered target	Enzymatic alteration of ribosomal target reduces drug binding	Various streptococci and staphylococci

Figure 11-9 Mode of β-lactamase enzyme activity. The enzyme cleaves the β-lactam ring, and the molecule can no longer bind to penicillin-binding proteins (PBPs) and is no longer able to inhibit cell wall synthesis. (Modified from Salyers AA, Whitt DD, editors: *Bacterial pathogenesis: a molecular approach,* Washington, DC, 1994, ASM Press.)

Staphylococci are the gram-positive bacteria that most commonly produce beta-lactamase; approximately 90% or more of clinical isolates are resistant to penicillin as a result of enzyme production. Rare isolates of enterococci also produce β-lactamase. Gram-negative bacteria, including Enterobacteriaceae, P. aeruginosa, and Acinetobacter spp., produce dozens of different β-lactamase types that mediate resistance to one or more of the β-lactam antibiotics.

Although the basic mechanism for β-lactamase activity shown in Figure 11-9 is the same for all types of these enzymes, there are distinct differences. For example, β-lactamases produced by gram-positive bacteria, such as staphylococci, are excreted into the surrounding environment, where the hydrolysis of β-lactams takes place before the drug can bind to PBPs in the cell membrane (Figure 11-10). In contrast, β-lactamases produced by gram-negative bacteria remain intracellular, in the periplasmic space, where they are strategically positioned to hydrolyze beta-lactams as they traverse the outer membrane through water-filled, protein-lined porin channels (see Figure 11-10). β-lactamases also vary in their spectrum of substrates; that is, not all β-lactams are susceptible to hydrolysis by every β-lactamase. For example, staphylococcal β-lactamase can readily hydrolyze penicillin and penicillin derivatives (e.g., ampicillin, mezlocillin, and piperacillin); however, it cannot effectively hydrolyze many cephalosporins or imipenem.

Figure 11-10 Diagrammatic summary of β-lactam resistance mechanisms for gram-positive and gram-negative bacteria. A, Among gram-positive bacteria, resistance is mediated by β-lactamase production and altered PBP targets. B, In gram-negative bacteria, resistance can also be mediated by decreased uptake through the outer membrane porins.

Various molecular alterations in the β-lactam structure have been developed to protect the β-lactam ring against enzymatic hydrolysis. This development has resulted in the production of more effective antibiotics in this class. For example, methicillin and the closely related agents oxacillin and nafcillin are molecular derivatives of penicillin that by the nature of their structure are not susceptible to staphylococcal β-lactamases. These agents are the mainstay of antistaphylococcal therapy. Similar strategies have been applied to develop penicillins and cephalosporins that are more resistant to the variety of β-lactamases produced by gram-negative bacilli. Even with this strategy, it is important to note that among common gram-negative bacilli (e.g., Enterobacteriaceae, P. aeruginosa, and Acinetobacter spp.), the list of molecular types and numbers of β-lactamases continues to emerge and diverge, thus challenging the effectiveness of currently available β-lactam agents.

Another therapeutic strategy has been to combine two different β-lactam moieties. One of the β-lactams (the β-lactamase inhibitor) has little or no antibacterial activity but avidly and irreversibly binds to the β-lactamase, rendering the enzyme incapable of hydrolysis; the second β-lactam, which is susceptible to β-lactamase activity, exerts its antibacterial activity. Examples of β-lactam/β-lactamase inhibitor combinations include ampicillin/sulbactam, amoxicillin/clavulanic acid, and piperacillin/tazobactam.

Altered targets also play a key role in clinically relevant β-lactam resistance (see Table 11-4). Through this pathway the organism changes, or acquires from another organism, genes that encode altered cell wall–synthesizing enzymes (i.e., PBPs). These “new” PBPs continue their function even in the presence of a β-lactam antibiotic, usually because the beta-lactam lacks sufficient affinity for the altered PBP. This is the mechanism by which staphylococci are resistant to methicillin and all other β-lactams (e.g., cephalosporins and imipenem). Methicillin-resistant S. aureus produces an altered PBP called PBP2a. PBP2a is encoded by the gene mecA. Because of the decreased binding between β-lactam agents and PBP2a, cell wall synthesis proceeds. Therefore, strains exhibiting this mechanism of resistance must be challenged with a non–β-lactam agent, such as vancomycin, another cell wall–active agent. Changes in PBPs are also responsible for ampicillin resistance in Enterococcus faecium and in the widespread β-lactam resistance observed in S. pneumoniae and viridans streptococci.

Because gram-positive bacteria do not have outer membranes through which β-lactams must pass before reaching their PBP targets, decreased uptake is not a pathway for β-lactam resistance among these bacteria. However, diminished uptake can contribute significantly to β-lactam resistance seen in gram-negative bacteria (see Figure 11-10). Changes in the number or characteristics of the outer membrane porins through which β-lactams pass contribute to absolute resistance (e.g., P. aeruginosa resistance to imipenem). Additionally, porin changes combined with the presence of certain β-lactamases in the periplasmic space may result in clinically relevant levels of resistance.

Resistance to Glycopeptides

To date, acquired, high-level resistance to vancomycin has been commonly encountered among enterococci, rarely among staphylococci, and not at all among streptococci. The mechanism involves the production of altered cell wall precursors unable to bind vancomycin with sufficient avidity to allow inhibition of peptidoglycan-synthesizing enzymes. The altered targets are readily incorporated into the cell wall, allowing synthesis to progress (see Table 11-4). A second mechanism of resistance to glycopeptides, described only among staphylococci to date, results in a lower level of resistance; this mechanism is thought to be mediated by overproduction of the peptidoglycan layer, resulting in excessive binding of the glycopeptide molecule and diminished ability of the drug to exert its antibacterial effect.

Because enterococci have high-level vancomycin resistance genes and also the ability to exchange genetic information, the potential for spread of vancomycin resistance to other gram-positive genera poses a serious threat to public health. In fact, the emergence of vancomycin-resistant S. aureus clinical isolates has been documented. In all instances the patients were previously infected or colonized with enterococci. Resistance to vancomycin by enzymatic modification or destruction has not been described.

Resistance to Aminoglycosides

Analogous to beta-lactam resistance, aminoglycoside resistance is accomplished by enzymatic, altered target, or decreased uptake pathways (see Table 11-4). Gram-positive and gram-negative bacteria produce several different aminoglycoside-modifying enzymes. Three general types of enzymes catalyze one of the following modifications of an aminoglycoside molecule (see Figure 11-4):

• Phosphorylation of hydroxyl groups

• Adenylation of hydroxyl groups

• Acetylation of amine groups

Once an aminoglycoside has been modified, its affinity for binding to the 30S ribosomal subunit may be sufficiently diminished or totally lost, allowing protein synthesis to occur.

Aminoglycosides enter the gram-negative cell by passing through outer membrane porin channels. Therefore, porin alterations may also contribute to aminoglycoside resistance among these bacteria. Although some mutations that resulted in altered ribosomal targets have been described, this mechanism of resistance is rare in bacteria exposed to commonly used aminoglycosides.

Resistance to Quinolones

Enzymatic degradation or alteration of quinolones has not been fully described as a key pathway for resistance. Resistance is most frequently mediated either by a decrease in uptake or in accumulation or by production of an altered target (see Table 11-4). Components of the gram-negative cellular envelope can limit quinolone access to the cell’s interior location where DNA processing occurs. Other bacteria, notably staphylococci, exhibit a mechanism by which the drug is “pumped” out of the cell, thus keeping the intracellular quinolone concentration sufficiently low to allow DNA processing to continue relatively unaffected. This “efflux” process, therefore, is a pathway of diminished accumulation of drug rather than of diminished uptake.

The primary quinolone resistance pathway involves mutational changes in the targeted subunits of the DNA gyrase. With a sufficient number or substantial major changes in molecular structure, the gyrase no longer binds quinolones, so DNA processing is able to continue.

Resistance to Other Antimicrobial Agents

Bacterial resistance mechanisms for other antimicrobial agents involve modifications or derivations of the recurring pathway strategies of enzymatic activity, altered target, or decreased uptake (Box 11-2).

Emergence and Dissemination of Antimicrobial Resistance

The resistance pathways that have been discussed are not necessarily new mechanisms that have recently evolved among bacteria. By definition, antibiotics originate from microorganisms. Therefore, antibiotic resistance mechanisms have always been part of the evolution of bacteria as a means of survival among antibiotic-producing competitors. However, with the introduction of antibiotics into medical practice, clinically relevant bacteria have adopted resistance mechanisms as part of their survival strategy. As a result of the increased use of antimicrobial agents, a survival of the fittest strategy has been documented as bacteria adapt to the pressures of antimicrobial attack (Figure 11-11).

All bacterial resistance strategies are encoded on one or more genes. These resistance genes are readily shared between strains of the same species, between species of different genera, and even between more distantly related bacteria. When a resistance mechanism arises, either by mutation or gene transfer, in a particular bacterial strain or species, it is possible for this mechanism to be passed on to other organisms using commonly described paths of genetic communication (see Figure 2-10). Therefore, resistance may spread to a wide variety of clinically relevant bacteria, and any single organism may acquire multiple genes and become resistant to the full spectrum of available antimicrobial agents. For example, strains of enterococci and P. aeruginosa already exist for which there are few effective therapeutic choices. Also, a gene encoding a single, very potent resistance mechanism may mediate multiple resistances. One such example is the mecA gene, which encodes staphylococcal resistance to methicillin and to all other beta-lactams currently available for use against these organisms; this leaves vancomycin as the only available and effective cell wall–inhibiting agent.

In summary, antibiotic use, coupled with the formidable repertoire bacteria have for thwarting antimicrobial activity and their ability to genetically share these strategies, drives the ongoing process of resistance emergence and dissemination (see Figure 11-11). This has been manifested by the emergence of new genes of unknown origin (e.g., methicillin-resistant staphylococci and vancomycin-resistant enterococci), the movement of old genes into new bacterial hosts (e.g., penicillin-resistant N. gonorrhoeae [PPNG]), mutations in familiar resistance genes that result in greater potency (e.g., beta-lactamase–mediated resistance to cephalosporins in Escherichia coli), and the emergence of new pathogens for which the most evident virulence factor is intrinsic or natural resistance to many of the antimicrobial agents used in the hospital setting (e.g., Stenotrophomonas maltophilia).

Because of the ongoing nature of the emergence and dissemination of resistance, reliable laboratory procedures to detect drug resistance serve as crucial aids to managing patients’ infections and as a means of monitoring changing resistance trends among clinically relevant bacteria.

Case Study 11-1

A 40-year-old Michigan resident with diabetes, peripheral vascular disease, and chronic renal failure was receiving dialysis. The previous history was significant for multiple courses of antimicrobial therapy, including vancomycin, for the treatment of a chronic foot ulcer and methicillin-resistant Staphylococcus aureus (MRSA) bacteremia. In June 2002, a culture of the dialysis catheter site demonstrated growth of S. aureus. The isolate was resistant to oxacillin (minimum inhibitory concentration [MIC] greater than16 µg/mL) and vancomycin (MIC greater than 128 µg/mL). A subsequent culture from the chronic foot ulcer revealed vancomycin-resistant S. aureus, vancomycin-resistant Enterococcus faecalis, and Klebsiella oxytoca.

Questions

1. Should the S. aureus isolate from the dialysis catheter site be reported as methicillin resistant?

2. What is the most likely mechanism by which the S. aureus isolate from the dialysis catheter site became resistant to vancomycin?