Antimicrobial agents are among the most commonly used and misused of all drugs. The inevitable consequence of the widespread use of antimicrobial agents has been the emergence of antibiotic-resistant pathogens, fueling an ever-increasing need for new drugs. However, the pace of antimicrobial drug development has slowed dramatically, with only a handful of new agents, few of which are novel, being introduced into clinical practice each year. Reducing inappropriate antibiotic use is thought to be the best way to control resistance. Although awareness of the consequences of antibiotic misuse is increasing, overprescribing remains widespread, driven largely by patient demand, time pressure on clinicians, and diagnostic uncertainty. If the gains in the treatment of infectious diseases are to be preserved, clinicians must be wiser and more selective in the use of antimicrobial agents.

Definition and Characteristics

In the strictest sense, antibiotics are antibacterial substances produced by various species of microorganisms (bacteria, fungi, and actinomycetes) that suppress the growth of other microorganisms. Common usage often extends the term antibiotics to include synthetic antimicrobial agents, such as sulfonamides and quinolones. Antibiotics differ markedly in physical, chemical, and pharmacological properties, in antimicrobial spectra, and in mechanisms of action. Knowledge of molecular mechanisms of bacterial replication has greatly facilitated rational development of compounds that can interfere with their replication.

Classification and Mechanism of Action

Antimicrobial agents are classified based on chemical structure and proposed mechanism of action, as follows: (1) agents that inhibit synthesis of bacterial cell walls, including the ?-lactam class (e.g., penicillins, cephalosporins, and carbapenems) and dissimilar agents such as cycloserine, vancomycin, and bacitracin; (2) agents that act directly on the cell membrane of the microorganism, increasing permeability and leading to leakage of intracellular compounds, including detergents such as polymyxin; polyene antifungal agents (e.g., nystatin and amphotericin B) which bind to cell-wall sterols; and the lipopeptide daptomycin (Carpenter and Chambers, 2004); (3) agents that disrupt function of 30S or 50S ribosomal subunits to reversibly inhibit protein synthesis, which generally are bacteriostatic (e.g., chloramphenicol, the tetracyclines, erythromycin, clindamycin, streptogramins, and linezolid); (4) agents that bind to the 30S ribosomal subunit and alter protein synthesis, which generally are bactericidal (e.g., the aminoglycosides); (5) agents that affect bacterial nucleic acid metabolism, such as the rifamycins (e.g., rifampin and rifabutin), which inhibit RNA polymerase, and the quinolones, which inhibit topoisomerases; and (6) the antimetabolites, including trimethoprim and the sulfonamides, which block essential enzymes of folate metabolism. There are several classes of antiviral agents, including: (1) nucleic acid analogs, such as acyclovir or ganciclovir, which selectively inhibit viral DNA polymerase, and zidovudine or lamivudine, which inhibit HIV reverse transcriptase; (2) non-nucleoside HIV reverse transcriptase inhibitors, such as nevirapine or efavirenz; (3) inhibitors of other essential viral enzymes, e.g., inhibitors of HIV protease or influenza neuraminidase; and (4) fusion inhibitors such as enfuvirtide . Additional categories likely will emerge as more complex mechanisms are elucidated. The precise mechanism of action of some antimicrobial agents still is unknown.

Factors That Determine the Susceptibility and Resistance of Microorganisms to Antimicrobial Agents

Successful antimicrobial therapy of an infection ultimately depends on the concentration of antibiotic at the site of infection. This concentration must be sufficient to inhibit growth of the offending microorganism. If host defenses are intact and active, a minimum inhibitory effect, such as that provided by bacteriostatic agents (i.e., agents that interfere with growth or replication of the microorganism but do not kill it) may be sufficient. On the other hand, if host defenses are impaired, antibiotic-mediated killing (i.e., a bactericidal effect) may be required to eradicate the infection. The concentration of drug at the site of infection not only must inhibit the organism but also must remain below the level that is toxic to human cells. If this can be achieved, the microorganism is considered susceptible to the antibiotic. If an inhibitory or bactericidal concentration exceeds that which can be achieved safely in vivo, then the microorganism is considered resistant to that drug.

The achievable serum concentration for an antibiotic guides selection of the breakpoint for designating a microorganism as either susceptible or resistant by in vitro susceptibility testing. However, the concentration at the site of infection may be considerably lower than achievable serum concentrations (e.g., vitreous fluid of the eye or cerebrospinal fluid). Local factors (e.g., low pH, high protein concentration, and anaerobic conditions) also may impair drug activity. Thus, the drug may be only marginally effective or ineffective in such cases even though standardized in vitro tests would likely report the microorganism as “sensitive.” Conversely, concentrations of drug in urine may be much higher than those in plasma. Microorganisms that might otherwise be considered “resistant” may be eradicated when infection is limited to the urinary tract.

Bacterial Resistance to Antimicrobial Agents

The recent emergence of antibiotic resistance in bacterial pathogens, both nosocomially and in the community, is a very serious development that threatens the end of the antibiotic era. Today, more than 70% of the bacteria associated with hospital-acquired infections in the United States are resistant to one or more of the drugs previously used to treat them. Penicillin-resistant strains of pneumococci account for 50% or more of isolates in some European countries, and the proportion of such strains is rising in the United States. The worldwide emergence of Haemophilus and gonococci that produce ?-lactamase is a major therapeutic problem. Methicillin-resistant strains of Staphylococcus aureus are endemic in hospitals and are isolated increasingly from community-acquired infections (Naimi et al., 2003; Vandenesch et al., 2003). Multiple-drug-resistant strains of S. aureus with intermediate susceptibility to antibiotics and high-level resistance to vancomycin have been reported (Hiramatsu et al., 1997; Smith et al., 1999; Weigel et al., 2003). There now are strains of enterococci, Pseudomonas, and Enterobacter that are resistant to all available antibiotics. Epidemics of multiple-drug-resistant strains of Mycobacterium tuberculosis have been reported in the United States.

The rampant spread of antibiotic resistance mandates a more responsible approach to antibiotic use. The Centers for Disease Control and Prevention has outlined a series of steps to prevent or diminish antimicrobial resistance. Important components include appropriate use of vaccination, judicious use and proper attention to indwelling catheters, early involvement of infectious disease experts, choosing antibiotic therapy based on local patterns of susceptibilities of organisms, proper antiseptic technique to ensure infection rather than contamination, appropriate use of prophylactic antibiotics in surgical procedures, infection control procedures to isolate the pathogen, and strict compliance to hand hygiene.

For an antibiotic to be effective, it must reach its target in an active form, bind to the target, and interfere with its function. Accordingly, bacterial resistance to an antimicrobial agent is attributable to three general mechanisms: (1) The drug does not reach its target, (2) the drug is not active, or (3) the target is altered (Davies, 1994; Spratt, 1994; Li and Nikaido, 2004).

The outer membrane of gram-negative bacteria is a permeable barrier that excludes large polar molecules from entering the cell. Small polar molecules, including many antibiotics, enter the cell through protein channels called porins. Absence of, mutation in, or loss of a favored porin channel can slow the rate of drug entry into a cell or prevent entry altogether, effectively reducing drug concentration at the target site. If the target is intracellular and the drug requires active transport across the cell membrane, a mutation or phenotypic change that shuts down this transport mechanism can confer resistance. For example, gentamicin, which targets the ribosome, is actively transported across the cell membrane using energy provided by the membrane electrochemical gradient. This gradient is generated by respiratory enzymes that couple electron transport and oxidative phosphorylation. A mutation in an enzyme in this pathway or anaerobic conditions (oxygen is the terminal electron acceptor of this pathway, and its absence reduces the membrane potential energy) slows entry of gentamicin into the cell, resulting in resistance. Bacteria also have efflux pumps that can transport drugs out of the cell. Resistance to numerous drugs, including tetracycline, chloramphenicol, fluoroquinolones, macrolides, and ?-lactam antibiotics, is mediated by an efflux pump mechanism (Li and Nikaido, 2004). Figure 42-1 depicts the multiple membrane and periplasm components that reduce the intracellular concentrations of ?-lactam antibiotics and cause resistance.

Drug inactivation is the second general mechanism of drug resistance. Bacterial resistance to aminoglycosides and to beta-lactam antibiotics usually is due to production of an aminoglycoside-modifying enzyme or beta-lactamase, respectively. A variation of this mechanism is failure of the bacterial cell to activate a prodrug. This is the basis of the most common type of resistance to isoniazid in M. tuberculosis (Bertrand et al., 2004).

The third general mechanism of drug resistance is target alteration. This may be due to mutation of the natural target (e.g., fluoroquinolone resistance), target modification (e.g., ribosomal protection type of resistance to macrolides and tetracyclines), or acquisition of a resistant form of the native, susceptible target (e.g., staphylococcal methicillin resistance caused by production of a low-affinity penicillin-binding protein) (Nakajima, 1999; Hooper, 2002; Lim and Strynadka, 2002).

Drug resistance may be acquired by mutation and selection, with passage of the trait vertically to daughter cells. For mutation and selection to be successful in generating resistance, the mutation cannot be lethal and should not appreciably alter virulence. For the trait to be passed on, the original mutant or its progeny also must disseminate and replicate; otherwise, the mutation will be lost until it is “rediscovered” by some other mutant arising from within a wild-type population.

Drug resistance more commonly is acquired by horizontal transfer of resistance determinants from a donor cell, often of another bacterial species, by transduction, transformation, or conjugation. Resistance acquired by horizontal transfer can disseminate rapidly and widely either by clonal spread of the resistant strain or by subsequent transfers to other susceptible recipient strains. For example, the plasmid-encoded staphylococcal ?-lactamase gene is distributed widely among many unrelated strains, including enterococci (Murray, 1992). Plasmid-encoded class A ?-lactamases of gram-negative bacteria also have spread widely to Escherichia coli, Neisseria gonorrhoeae, and Haemophilus spp. Horizontal transfer of resistance offers several advantages over mutation-selection. Lethal mutation of an essential gene is avoided; the level of resistance often is higher than that produced by mutation, which tends to yield incremental changes; the gene, which still can be transmitted vertically, can be mobilized and rapidly amplified within a population by transfer to susceptible cells; and the resistance gene can be eliminated when it no longer offers a selective advantage.

Mutation-Selection

Mutation and antibiotic selection of the resistant mutant are the molecular basis for resistance to streptomycin (ribosomal mutation), quinolones (gyrase or topoisomerase IV gene mutation), rifampin (RNA polymerase gene mutation), and linezolid (ribosomal RNA mutation). This mechanism underlies all drug resistance in M. tuberculosis (Riska et al., 2000). Mutations may occur in the gene encoding (1) the target protein, altering its structure so that it no longer binds the drug; (2) a protein involved in drug transport; (3) a protein important for drug activation or inactivation, in the case of extended-spectrum beta-lactamases (Bush, 2001); or (4) in a regulatory gene or promoter affecting expression of the target, a transport protein, or an inactivating enzyme. Mutations are not caused by drug exposure per se. They are random events that confer a survival advantage when drug is present. However, certain drugs that induce the bacterial SOS system of DNA repair proteins that accommodate potentially lethal stress (e.g., fluoroquinolones) may facilitate resistance gene transfer or increase the mutation frequency by induction of error-prone polymerases (Goodman, 2002; Chopra et al., 2003; Beaber et al., 2004). Any large population of antibiotic-susceptible bacteria is likely to contain rare mutants that are only slightly less susceptible than the parent. Through sequential acquisition of more mutations, clinically significant resistance may emerge. High-level resistance of E. coli to fluoroquinolones is due to such an accumulation of multiple stepwise mutations. In some instances, a single-step mutation results in a high degree of resistance. For example, a point mutation within the drug-binding domain in the ? subunit of bacterial RNA polymerase confers high-level resistance to rifampin.

Horizontal Gene Transfer

Horizontal transfer of resistance genes is greatly facilitated by and is largely dependent on mobile genetic elements. The role of plasmids and transducing phages as carriers of resistance genes and transfer elements is discussed in more detail below. Other mobile elements, transposable elements, integrons, and gene cassettes also participate in the process. Transposable elements are of three general types: insertion sequences, transposons, and transposable phages; two of these, insertion sequences and transposons, are important for resistance. Insertion sequences (Mahillon and Chandler, 1998) are short segments of DNA encoding enzymatic functions (e.g., transposase and resolvase) for site-specific recombination with inverted repeat sequences at either end. They can copy themselves and insert themselves into the chromosome or a plasmid. Insertion sequences do not encode resistance, but they function as sites for integration of other resistance-encoding elements, e.g., plasmids or transposons.

Transposons are basically insertion sequences that also code for other functions, one of which can be drug resistance. Since transposons move between chromosome and plasmid, the resistance gene can hitchhike its way onto a transferable element out of the host and into a recipient. Transposons are mobile elements that excise and integrate in the bacterial genomic or plasmid DNA (i.e., from plasmid to plasmid, from plasmid to chromosome, or from chromosome to plasmid).

Integrons (Fluit and Schmitz, 2004) are not formally mobile and do not copy themselves, but they encode an integrase and provide a specific site into which mobile gene cassettes integrate. Gene cassettes encode resistance determinants, usually lacking a promoter, with a downstream repeat sequence. The integrase recognizes this repeat sequence and directs insertion of the cassette into position behind a strong promoter that is present on the integron. Integrons may be located within transposons or in plasmids, and therefore may be mobilizable, or located on the chromosome.

Another type of gene cassette, SCCmec (Staphylococcal Chromosomal Cassette), has been described in methicillin-resistant strains of staphylococci (Katayama et al., 2000). The methicillin resistance gene mecA is located within this cassette along with recombinase genes. The recombinases both excise and integrate the cassette element, which exists as a circular intermediate that is not self-replicating, into a very specific site in the staphylococcal chromosome. How this element is transferred and the role of excision-mobilization in this process are not known.

Transduction

Transduction is acquisition of bacterial DNA from a phage (a virus that propagates in bacteria) that has incorporated DNA from a previous host bacterium within its outer protein coat. If the DNA includes a gene for drug resistance, the newly infected bacterial cell may acquire resistance. Transduction is particularly important in the transfer of antibiotic resistance among strains of S. aureus.

Transformation

Transformation is the uptake and incorporation into the host genome by homologous recombination of free DNA released into the environment by other bacterial cells. Transformation is the molecular basis of penicillin resistance in pneumococci and Neisseria (Spratt, 1994). Penicillin-resistant pneumococci produce altered penicillin-binding proteins (PBPs) that have low-affinity binding of penicillin. Nucleotide sequence analysis of the genes encoding these altered PBPs indicates that they are mosaics in which blocks of foreign DNA from a closely related species of streptococcus have been imported and incorporated into the resident PBP gene.

Conjugation

Conjugation is gene transfer by direct cell-to-cell contact through a sex pilus or bridge. This complex and fascinating mechanism for the spread of antibiotic resistance is extremely important because multiple resistance genes can be transferred in a single event. The transferable genetic material consists of two different sets of plasmid-encoded genes that may be on the same or different plasmids. One set encodes the actual resistance; the second encodes genes necessary for the bacterial conjugation process.

Conjugative plasmids tend to be rather large (50 kilobases or more). They combine elements of plasmid DNA rolling-circle replication (only a single strand is transferred, and it replicates in the host) with a type IV bacterial secretion system. Plasmid transfer requires an origin of transfer demarcating the site within the plasmid where transfer will occur, DNA replicating enzymes, and coupling proteins that direct the DNA across two cell membranes on its way from the host into the recipient. Genes encoding the resistance determinants may be located on transposons.

Conjugation with genetic exchange between nonpathogenic and pathogenic microorganisms probably occurs in the GI tracts of human beings and animals. The efficiency of transfer is low; however, antibiotics can exert a powerful selective pressure to allow emergence of the resistant strain. Genetic transfer by conjugation is common among gram-negative bacilli, and resistance is conferred on a susceptible cell as a single event. Enterococci also contain a broad range of host-range conjugative plasmids that are involved in the transfer and spread of resistance genes among gram-positive organisms. Vancomycin resistance in enterococci is mediated by a conjugative plasmid . Vancomycin resistance in S. aureus is due to conjugative transfer of vanA-type vancomycin resistance genes encoded on a transposon from Enterococcus faecalis donor into a methicillin-resistant strain of S. aureus with subsequent integration of the transposon into a resident staphylococcal conjugative plasmid.

Source: GOODMAN & GILMAN’S THE PHARMACOLOGICAL BASIS OF THERAPEUTICS

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