Charles William Stratton
Importance of Understanding the Molecular Basis of Antimicrobial Action
Antimicrobial action can be defined as the interaction of the drug with the microorganism; this interaction is widely described as antimicrobial pharmacodynamics (1). The result of this interaction of the drug with the microorganism may be inhibition or death of the microbe or may instead be the emergence of a resistant subpopulation of microorganisms that can negate the effect of the antibiotic (2–5). Pharmacokinetics, on the other hand, is the interaction of the drug with the patient (6). The integration of pharmacodynamics with pharmacokinetics in the first decade of the 21st century has greatly improved optimal antimicrobial therapy (6–13). Integral to the application of pharmacodynamic and pharmacokinetic principles to antimicrobial therapy is an understanding of the molecular basis of antimicrobial action. Such an understanding begins with an understanding of the molecular targets for antimicrobial agents. These molecular targets include microbial RNA and DNA; microbial biosynthesis of proteins, folic acid, and cell wall (peptidoglycan); microbial membrane function; and microbial energy metabolism. The specific interaction of antimicrobial agents with each of these molecular targets must be understood in order to optimally use pharmacodynamic and pharmacokinetic principles (14).
Although the global emergence of resistance may well be an inevitable and unavoidable result of the use of antimicrobial agents (3,15,16), the understanding and application of the molecular basis of antimicrobial action can minimize the degree of resistance that occurs (15–18). Moreover, understanding the molecular basis of antimicrobial action allows better appreciation of resistance mechanisms, which, in turn, allows these resistance mechanisms themselves to be targeted by antimicrobial agents (19). Moreover, understanding of the molecular targets provides a more rational approach for the use of combination antimicrobial therapy (20–23). The therapy of tuberculosis (TB) serves as an example of such a rational approach. Here, the use of combination therapy with new and/or different classes of antimycobacterial agents (24,25) that specifically target the dormant phase of mycobacteria has resulted in improved efficacy, whereas the use of specific therapeutic approaches such as directly observed therapy (26) has resulted in lower rates of resistance. It is therefore the purpose of this chapter to first review general principles that are useful for understanding the molecular basis of antimicrobial action and then to review mechanisms of action for selected classes of antimicrobial agents in current use or under investigation.
GENERAL PRINCIPLES OF ANTIMICROBIAL ACTION
Antimicrobial Mechanisms of Action in Relationship to the Cellular Structure and Physiology of Microbes
Importance of Microbial Physiology in Antimicrobial Action
The importance of the microbial growth phase for the in vitro effects of antimicrobial action has long been appreciated in clinical microbiology (27–31). However, this is only a small part of the microbial physiology that must be understood in order to optimally use antimicrobial agents. Microbial physiology is a complex subject with important implications for the effectiveness of antimicrobial agents, implications that have only recently become understood. Fortunately, there has been a great deal of progress made in understanding this microbial physiology and its influence on antimicrobial action. It is important to appreciate that microorganisms have mechanisms for replication, including synthesis of the cell wall (32–35), an important target of antimicrobial therapy (36,37). In addition to synthesis of the cell wall, a microorganism must also be able to focally lyse its cell wall so that replication can occur (33,36,38–40). The enzymes that mediate lysis of the cell wall must be tightly controlled; otherwise, they could cause destruction of the cell. These enzymes are preformed and are diffusely present in the cell wall until focally activated (39,40). Moreover, these enzymes can be globally activated as part of programmed cell death (i.e., apoptosis) or can be triggered by antimicrobial agents (41–45). Furthermore, microorganisms have evolved compensatory mechanisms to deal with times of starvation; these mechanisms ensure that the genome of some microorganisms survive until better times (46–48). Such mechanisms include quorum sensing, which may activate programmed cell death as the microbial population increases and nutrients become sparse (42,49,50). For example, programmed cell death in bacteria is a major factor in the development of biofilm (49,51,52). Microorganisms also have repair mechanisms when subjected to DNA damage, a reaction known as the “SOS” response; failing repair, programmed cell death is likely to occur (53–55). Finally, microorganisms often live in microcolonies in biofilm, and the behavior of a cell is greatly influenced by the location of the cell within the microcolony (52,56–58). Into this complex physiologic environment, antimicrobial agents are introduced. Attempting to understand the mechanism of action of these agents without understanding microbial physiology is futile. Accordingly, microbial physiology will be discussed in some detail.
Role of the Physiologic State of the Microbe in Antimicrobial Activity
The specific physiologic state of the microorganism, particularly its surface properties and rate of cell replication, markedly influences the activity of antimicrobial agents and partially determines whether they are bactericidal or bacteriostatic as well as their rate of killing (28–31,59–62). It has become clear that the pure in vitro broth culture, albeit the mainstay of antimicrobial susceptibility testing, is an artifact emphasizing free-floating mobile (planktonic) cells, which exist primarily in the laboratory setting and not in nature (i.e., infections) (29,62). This discriminates against the adherent (sessile) cells present in biofilm, which has now been acknowledged as the predominant growth form in natural ecosystems, including most infections (49,52,56–58,63). Moreover, microbial growth forms in biofilm have been associated with persistent infections and microbial resistance (49,60,63–65). Although noted long ago, it is no less true today: a solid support surface for the growth of bacteria provides a better approximation of the in vivo state than does broth medium (29).
Cellular Physiology of Microbial Life and Death
The growth of microorganisms in natural environments is characterized by periods of nutrient starvation, which results in growth rates that approximate zero (46–48,66). Nonetheless, bacteria are able to survive for prolonged periods of time despite the relative absence of nutrients. The survival of bacteria under these starvation conditions involves induction of a number of genes or proteins that mediate physiologic changes that enable the survival of some of the cells (46–48,66,67). These physiologic changes also can be seen as a microorganism transitions from the logarithmic phase of growth to the stationary phase, during which time the expression levels of a number of gene products produce marked phenotypic changes (31,66–69). Finally, these changes are seen after exposure of the microorganism to antimicrobial agents (70–72). The following examples are illustrative. Nutrient limitation in isolates of Pseudomonas aeruginosa has been shown to result in increased synthesis of exopolysaccharides (66,73,74). This phenotypic change, in turn, results in increased biofilm that appears to decrease the antimicrobial activity of a number of agents (75,76), including β-lactam agents such as piperacillin (77). Clinically, emergence of P. aeruginosa strains producing high levels of persister cells has been noted in patients with cystic fibrosis (65). Microbial stress can result in alteration of penicillin-binding proteins (PBPs) and cell walls. For example, heat shock protein ClpL in Streptococcus pneumoniae has been shown to induce a ClpL-dependent increase in the messenger RNA (mRNA) levels and protein synthesized by the cell wall synthesis gene pbp2x, which results in a thicker cell wall and higher resistance to penicillin (78). In response to a broad range of environmental stresses, wild-type strains of Escherichia colibecome short coccobacillary forms that exhibit increased levels of resistance (79). These morphologic changes in E. coli are associated with alterations in the expression of PBPs, with an increase seen in the amount of PBP6 and a decrease in PBP3 (80–82). Similar changes in the PBPs of Streptococcus pyogenes (31) and Haemophilus influenzae (68) during the stationary phase have been reported. This suggests some common effects for PBPs during the stationary phase on microorganisms. Lack of certain nutrients also may play a role (47,83,84). For example, limitation of choline in the teichoic acids of S. pneumoniae results in replacement by ethanolamine, which subsequently inhibits the growing cells from splitting into diplococci (85). All of these changes and more appear to be triggered by stress, stationary phase, and/or limitation of nutrients and to be essential for continued cell survival during prolonged periods in the stationary phase (47,48,66,67,69,81,86).
Among other important changes that occur under starvation conditions are those associated with the stringent response (30,87). The stringent response is an adaptation to conditions of amino acid starvation. This response includes induction of specific enzymes such as guanosine tetraphosphate (ppGpp) in the initial stage, which then increases the transcription of both inducible and repressible enzyme operons involved in the stringent response (30,84,87–89). Examples of the stringent response include phenotypic changes seen in marine bacterial cells (83) as well as the latent infection caused by Mycobacterium tuberculosis (89). These changes in some microorganisms are characterized by rapid multiple divisions of starved cells, leading to the formation of ultramicrobacteria (<0.3 µm in diameter), which are also called dwarf forms (90). Rapid formation of multiple copies is presumed to improve the chances of individual genomes surviving. These cells are dormant forms and are quite resistant to many antimicrobial agents as well as to osmotic stress.
Starvation conditions are not the only conditions that may induce the stringent response. Other conditions that appear to induce the stringent response are exposure to certain antibiotics, including β-lactam agents. Lorian (91–93) demonstrated that staphylococci, when exposed to subinhibitory concentrations of penicillin, produced abnormally large cells that were actually clusters of smaller dwarf staphylococci crowded together within a single surrounding thickened cell wall and prevented from separating by the presence of many wide cross-walls. Comparison of the ultrastructure of staphylococci following penicillin exposure with that of staphylococci isolated from osteomyelitis in animal models revealed morphologies that were virtually indistinguishable (94). When incubated on drug-free media, these clusters of staphylococci separated into smaller clusters and eventually become normal-sized individual cells. This morphogenesis and fatal variations in the presence of penicillin of the staphylococcal cell wall are reviewed in more detail by Giesbrecht et al. (95).
These dwarf forms of staphylococci crowded together within a single thickened surrounding cell wall are likely to be dormant forms produced by the stringent response due to limited cell wall damage caused by subinhibitory concentrations of β-lactam agents. In contrast, higher levels of β-lactam agents cause enough cell wall damage to activate autolytic mechanisms (44,45,96). These differences, thus, may simply reflect a difference in the physiologic response to cell wall damage: limited cell wall damage triggers a stringent response, whereas extensive damage triggers apoptosis.
Multicellular forms of staphylococci also are seen in mutant staphylococci that have had their scdA gene inactivated (97). This aberrant cellular morphology is similar to those of staphylococci carrying femAand femB mutations (98). This suggests that the transient changes described by Lorian and colleagues (91,92,93) may become permanent after mutation of specific genes involved in this cellular morphology. Multiple dwarf copies of a microorganism within one thickened cell wall would presumably be more resistant to antimicrobial agents or other noxious substances and also would increase the chance of the genome surviving.
Similar effects have been described for other microorganisms. Helicobacter pylori, for example, has been shown to produce coccoid forms, which have been attributed to environmental stress such as starvation (69,99). These coccoid forms have also been found to emerge after exposure to antibiotics such as amoxicillin (100). They are not culturable in vitro but revert to culturable forms in mice (101). In Bilophila wadsworthia, scanning and transmission electron microscopy have demonstrated that subinhibitory concentrations of imipenem result in large multilobate cells, suggesting that new growth of cells was initiated while cell division or separation was inhibited (102). A similar effect of imipenem has been reported for P. aeruginosa isolates, where exposure to concentrations of imipenem greater than the relevant minimal inhibitory concentrations (MICs) results in large spheroplasts with evidence of cross-wall formation just before lysis (103). This effect presumably extends to the newer carbapenems such as meropenem. Dwarf forms of P. aeruginosa have been observed in microcolonies within the lung tissue of patients with cystic fibrosis (90,104,105). Each of these aberrant forms described most likely represents the entry of the microorganism into the stringent response phase.
It is of interest that, while long-starved cells are resistant to cell wall–active agents as well as agents that inhibit DNA synthesis, they remain somewhat susceptible to certain agents that inhibit protein synthesis (106) or interfere with bacterial membrane function (107). Staphylococcus aureus exposed to subinhibitory concentrations of antimicrobial agents that interfere with protein synthesis develop a thick cell wall (94) with one or two thick cross-walls, except in the case of tetracycline, where no cross-wall formation has been observed (108). This older observation has greater significance with the more recent demonstration of successful in vivo therapy of methicillin-resistant Staphylococcus aureus (MRSA) endocarditis with minocycline in the clinical setting (109) as well as in an experimental endocarditis model (110). In this experimental endocarditis model, minocycline was as effective as vancomycin. Recognition of the bactericidal effects of minocycline on microorganisms in a dormant phase may ultimately result in an additional antimicrobial agent for the therapy of methicillin-resistant staphylococci (111). This observation is likely to become more important as the incidence of community-acquired MRSA increases (112,113).
Cellular Physiology of Microbial Apoptosis and Repair
There are a number of physical or chemical agents in the natural environment of microbes that can cause damage to cells (114). The most vulnerable portions of microbial cells are the cell wall/membrane (36,37,107) and the cell genome (115–121). Therefore, it is not surprising that microbial cells have evolved repair systems for these vulnerable targets. If the damage to the microbial cell is severe and cannot be repaired, an apoptosis (programmed cell death) mechanism is activated (41,42,53–55,122,123). Programmed cell death may, for example, play a role in the elimination of bacterial cells that are damaged by cell wall–active agents such as penicillins (42–45,95–97,124). The balance between repair and programmed cell death may well be an unappreciated target of antimicrobial therapy; it is likely that rapid killing of microbes involves activation of such apoptotic mechanisms (44,45). Indeed, a number of major classes of bactericidal antimicrobial agents have been shown to induce hydroxyl radical formation that is an end product of an oxidative damage cellular death pathway that, in turn, leads to microbial cell death (44,45,125). For example, amoxicillin therapy in a rabbit endocarditis model has been shown to kill Enterococcus faecalis by two mechanisms (126). The first is autolysin independent and likely corresponds to the production of reactive oxygen species (44,45). The second is autolysin dependent and involves the loss of the osmoprotective function of the peptidoglycan at a high cell density. A similar dual mechanism of killing for penicillin against S. pneumoniae has been described (127). Apoptotic mechanisms may also explain species-specific bactericidal activity. An example is seen with chloramphenicol, which kills certain species such as S. pneumoniae and H. influenzae but not Escherichia coli. This may simply reflect differences in the activation of apoptotic mechanisms.
Prevention of damage to vital portions of the microbial cell is clearly an important prelude to cellular repair. Accordingly, microbial cells have developed an effective protective barrier for their cell wall/membrane called biofilm(52,56,57). Biofilm is discussed in detail due to its importance in infections and as a future target for antimicrobial therapy (49,52,57,63,64,128). Repair of this biofilm and of the underlying cell wall/membrane structure when damage occurs is an important microbial function. Biofilm repair involves synthesis of the precursors within the cytoplasm, translocation of these precursors to the outer portions of the cell wall/membrane, and final assembly of the biofilm matrix. Studies have shown that biofilm repair is dependent on a carbohydrate source, an energy source, certain enzymes, and functioning efflux pumps. With these components available, repair occurs very quickly (129). There is now intriguing evidence that macrolides can interfere with the synthesis and repair of biofilm; this process seems to involve inhibition of quorum-sensing as well as the elaboration of exotoxins by means of codon–anticodon interactions that inhibit the translation of mRNA for inducible enzymes (130–138). This important topic is also discussed in detail.
Repair of any damage to the cell wall/membrane of microbes appears to be most easily accomplished by replication, provided the damage is not extensive. This may be an important factor in combination antimicrobial therapy specifically directed at creating functional synergy of two antimicrobial agents against an infectious microbe. Such an approach, for example, might involve the use of cell wall disrupters combined with agents active against replicating microorganisms.
The repair of structural damage to DNA is also of considerable importance to the microbe, because this damage (and the repair) might result in mutations that could be lethal. Accordingly, the response to DNA damage by the microorganism is complex (53–55). There are three important mechanisms of DNA repair in microorganisms: (a) direct repair, which restores the original structure; (b) indirect repair, in which one DNA strand is bypassed during replication or is excised and then rebuilt by copying the intact strand; and (c) postreplication repair, in which the damage is eliminated by recombination between the sister strands after replication.
A major mechanism for indirect repair of damage that blocks chain elongation during replication is that provided by the SOS system (53–55,139). The SOS system is a set of approximately 20 damage-inducible (din) genes. The SOS response is controlled by two regulatory proteins, which are the products of the lexA and recA genes. The first protein product of the gene lexA normally represses the SOS response. Upon SOS induction, the recA gene produces RecA protein. Damaged DNA, in the presence of the single-strand–binding protein, binds RecA protein in a way that changes its configuration so that it becomes a protease that cleaves lexA. This results in derepression of the other genes. The induction of RecA protein can be inhibited by antimicrobial agents such as chloramphenicol, erythromycin, and tetracycline, which have codon–anticodon interactions that inhibit the translation of mRNA needed for the synthesis of inducible enzymes.
Once activated, the SOS response has several effects. One of these is induction of DNA polymerases, which will be needed when cell division resumes (140). Another is cell division inhibition mediated by sfiA and sfiC, which target the ftsZ gene and protein, important factors in cell separation (141). Overproduction of the ftsZ protein has been shown to produce ultramicrobacteria. The SOS response is primarily involved in DNA repair (54,55). The major mechanism of repair of the activated SOS system is bypass repair. This particular mechanism of DNA repair tends to be error-prone and often results in mutants (54,55,140,142).
Cell Wall/Biofilm Structures of Gram-Positive and Gram-Negative Microorganisms
The cell walls of both gram-positive (Fig. 10.1) and gram-negative (Fig. 10.2) bacteria are similar, in that both possess inner cytoplasmic membranes as well as outer peptidoglycan (murein) layers (33,35,143–145). Gram-negative bacteria also have an additional outer cell membrane, which covers the peptidoglycan layer (82,143,145–147). Finally, the cell walls of both gram-positive and gram-negative bacteria interface with the external milieu via biofilm, which is a matrix-supported gel (52,56). Each of these structures plays a critical role in the interaction of antimicrobial agents with the microorganism (36,37,107,128).
The cytoplasmic membrane in each group of bacteria is a semipermeable membrane that regulates molecular flow, in turn determining pH (143,148,149), osmotic pressures (150), and availability of essential substances. The peptidoglycan layer in each group is a continuous cross-linked mesh that forms a polyionic and amphoteric network (33,34,143). The peptidoglycan mesh is composed of linear glycan chains that are interlinked by short peptides (33,34,82,143,144,151). This shell surrounds the entire microorganism, is known as a sacculus, and is found exclusively in eubacteria. The peptidoglycan sacculus is not a rigid shell but instead is elastic and flexible. This relatively porous peptidoglycan sacculus (exclusion limits of 100,000 Da) serves as a mechanical “exoskeleton” that helps to maintain the microorganism’s shape, rigidity, and osmotic stability. The exoskeleton of gram-positive bacteria is thicker than that of gram-negatives, thus providing more rigidity. Although this polyanionic sacculus might appear to be an exclusion barrier, the exclusion limits of 100,000 Da make this meshwork very coarse and thus allow molecules of lesser size, such as antimicrobial agents, which have sizes of 300 to 700 Da, to readily diffuse through the layer. Finally, the peptidoglycan structure is involved in the cell division process (32,34,152).
The molecular structures of the cytoplasmic membranes of gram-positive and gram-negative bacteria are essentially the same, consisting of lipid bilayers containing phospholipids and membrane proteins (143–145). There are, however, important differences in the peptidoglycan wall and the biofilm for these two groups of bacteria. Gram-positive bacteria have a relatively simple but thick cell wall constructed of peptidoglycan and teichoic acids, which are long-chain polymers consisting of glycerol or ribitol residues with phosphodiester links and various substituents such as uronic acids (85,144,153). Teichoic acids are found as either cell-bound or free soluble acids. The cell wall of gram-positive bacteria contains two forms of the cell-bound teichoic acid. In one form, lipoteichoic acid, one end of the chain is anchored to phospholipids in the cytoplasmic membrane while the other end transverses the peptidoglycan layer in such a way that it protrudes at the cell surface (153). In the second, a cell wall teichoic acid, one end is attached to N-acetylmuramyl residues in the peptidoglycan layer and the free end protrudes at the cell surface. Finally, free, soluble teichoic acid is present in large amounts in the outer portion of the cell wall.
At the surface of the gram-positive cell wall, the protruding teichoic acids can be linked with one another via branching polysaccharides to form a matrix. The cross-linking of the biofilm matrix is accomplished using polysaccharides with repeating units of two or three sugars. The variety of possible hexose stereoisomers and of linkages, as well as the potential incorporation of unusual sugar residues, results in thousands of different trisaccharides in the matrix. Because the polysaccharide chains in the matrix are hydrophilic, water is absorbed into the matrix and transforms this outer layer into a gel. This matrix-supported gel (99% water) is known by a variety of names, including biofilm, glycocalyx, slime, alginate, and capsule (52,56).
One of the key components of the biofilm is phospholipids because of the covalent bonding they provide. In addition, this hydrated matrix depends on calcium and magnesium cations to maintain the negatively charged ends of the polysaccharides in close approximation. The availability of phospholipids and divalent cations in the medium greatly influences the final composition of the cell wall. This can be appreciated by considering the gram-positive cell wall. Under conditions of magnesium limitation, gram-positive bacteria increase the amount of teichoic acid produced while decreasing the amount of alanyl ester substitutions, which results in fewer polysaccharides present in the matrix and hence less biofilm. Phosphate limitation results in the replacement of teichoic acids by teichuronic acids, which have less covalent bonding. Starvation conditions, then, would be predicted to result in cell walls with minimal biofilm.
The cell walls of gram-negative bacteria differ from those of gram-positive bacteria in a number of ways (82,147,154). First, they have a relatively thin peptidoglycan layer, which provides less rigidity. Gram-negative cell walls also have an additional outer cell membrane (145–147) that serves as an effective permeability barrier (155–157). This outer cell membrane is composed of two layers. The inner layer consists largely of glycerophospholipid molecules with two covalently linked fatty acid chains, a molecular structure that is fairly common in membranes in general. The outer layer is somewhat unique among membranes and contains mainly two classes of proteins, lipoproteins and β-barrel proteins. Lipoproteins contain lipid molecules that are coupled with an amino-terminal cystein, whereas β-barrel proteins are β sheets wrapped into cylinders and referred to as outer membrane proteins (OMPs). The outer membrane also contains glycopeptides, principally lipopolysaccharides with six or seven covalently linked fatty acid chains. The fatty acids present in the lipopolysaccharides are saturated. This results in the interior of the lipid bilayer being less fluid, because there is no packing of carbons, as seen when the fatty acids are unsaturated (156). This serves to make this barrier more restrictive to hydrophilic agents.
Areas of adhesion between the outer cell membrane and the inner cytoplasmic membrane, called Bayer’s junctions, have been described by a number of investigators. These adhesions of the inner cytoplasmic membrane to the cell peptidoglycan/outer membrane are probable sites of synthesis of the outer membrane and biofilm as well as synthesis of other substrates to be pumped from the cell cytoplasm into the periplasmic space or from the cell cytoplasm directly into the biofilm.
The periplasmic space is an aqueous cellular compartment delineated by the outer cell membrane and the inner cytoplasmic membrane (158). The periplasmic space contains various proteins that are densely packed, making this aqueous milieu more viscous than the cytoplasm. These proteins include potentially harmful proteins such as RNAse or alkaline phosphatase, thus making the periplasmic space an evolutionary precursor of the lysosomes of eukaryotic cells (159).
Gram-negative bacteria, like gram-positive bacteria, are surrounded by a polyanionic polysaccharide matrix, which differs mainly by being anchored by the lipooligosaccharides rather than by lipoteichoic acids. In some instances, the attachment is to the peptidoglycan layer, whereas in others the lipid A is anchored to the inner cytoplasmic membrane. This lipopolysaccharide matrix of gram-negative bacteria is also thought to provide some selectivity/hindrance via negative ionic charges and/or steric hindrance (160).
Biofilms of both gram-positive and gram-negative bacteria are essentially anionic polymeric diffusion barriers and can be thought of as an ion-exchange resin of almost infinite surface area. In addition, the biofilm protects the microorganism from heavy metal toxicity, from most bacteriophages, from phagocytic white blood cells, from antibodies and/or complement, and from an inhospitable milieu such as osmotic, pH, or enzymatic dangers. The interface of the biofilm with the aqueous phase can be altered by methylation of fatty acids or by sulfonation of polysaccharides, which increases the barrier to water-soluble agents (161). Bacteria are able to excrete into their biofilm and the surrounding medium several different classes of molecules, including exopolysaccharides (the building blocks of biofilm), siderophores, protein enzymes, and toxins (146). Biofilms also appear to serve as a repository for defensive substances such as β-lactamase (162).
In human infections, microbial cells are most often found with biofilm (52,57,58,63), even though microscopic examination may not readily reveal this. In clinical microbiology laboratories, the optimal growth conditions sought by microbiologists are far from the near-starvation circumstances the bacteria encounter in their natural environments. This is particularly true for broth media (29). However, the growth of gram-positive and gram-negative bacteria on agar plates in the laboratory may reflect, in part, the presence or absence of biofilm. Smooth colonies have more biofilm than do rough colonies, while mucoid or slimy colonies have the most. Deep rough colonies, on the other hand, have the smallest amount of biofilm, if any at all. These deep rough mutants have most of the core lipid A eliminated. Such strains are more susceptible to lysozyme and more permeable to hydrophobic antibiotics. Finally, in clinical microbiology laboratories, biofilm may be recognized and described microscopically as capsule.
Consequences of Biofilm Disruption
The disruption of the bacterial cell wall often results in the death of the microorganism (114). This is well appreciated by clinicians. Less well appreciated is that the disruption of biofilm surrounding an individual microbial cell is not without consequences (163–165). These may be related to osmotic pressure and the shifting of cell peptidoglycan by that pressure. There is a higher hydrostatic pressure within the cytoplasmic space of a microbe than that which is exerted on the cell by the external milieu. The presence of the biofilm matrix seems to assist in keeping this internal pressure in check (172). When a portion of the gel is removed, however, the internal pressure shifts the cell wall/membrane so that it protrudes through this disrupted area, resulting in a fingerlike projection containing cytoplasmic contents (164,166–168). Extrusion of this portion of the cell wall/membrane through the hole in the biofilm is a result of the cell wall/membrane shifting to adjust to the focused pressure directed at the area of disrupted biofilm. This shift in turn activates autolytic enzymes to dissolve the peptidoglycan component of the cell wall as the wall shifts during replication (169). This causes dissolution of cell wall peptidoglycan in this area, which, in conjunction with the high, focused internal pressure, effectively severs this protruding bleb (170), leaving a transient hole. If enough holes are formed, leakage of cytoplasmic contents results in cellular death (171). The results of this process can be demonstrated by electron microscopy, which makes visible a range of ultrastructural changes, including narrow fingerlike projections, blebs, and extracellular cytoplasmic-filled vesicles. Disruption of the entire biofilm matrix, in contrast, tends to equalize the pressure over the entire cell wall/membrane. Consequently, when the autolytic enzymes dissolve the peptidoglycan of the entire cell, the result may be lysis of the entire cell or the creation of a spheroplast if the osmolarity of the external milieu is sufficiently high. When lysis is seen, it occurs rapidly, in contrast to the lysis seen after exposure to penicillin, where cells continue to grow for approximately half a generation before lysis (27,170). Finally, it appears that gram-negative bacteria are more susceptible to the effects of biofilm disrupters, perhaps because of their less rigid cell walls.
Disruption of biofilm can be accomplished by a number of physicochemical mechanisms (161). This disruption can be best appreciated by electron microscopy. The ultrastructure of normal cells of gram-negative or gram-positive bacteria has a slightly undulating smooth surface, which is transformed to a surface with blebs and tubular projections after displacement of Ca2+ and Mg2+ from the biofilm. Displacement of these cations from the biofilm by chelating agents such as ethylenediaminetetraacetic acid (EDTA) (155,170) or by polycationic agents (164,172) such as polymyxin B (166,167) and aminoglycosides (173,174) has been shown to be an effective way to disrupt biofilm, although this mechanism can be countered by the presence of excessive amounts of calcium and magnesium cations in the milieu (175–177). However, adding these cations after the damage has been done has no effect. The ultrastructural changes seen by electron microscopy are accompanied by a functional change, namely, increased permeability to hydrophobic agents such as antibiotics (146,163,178).
If the changes induced by the disruption of biofilm are not rapidly fatal and the cells are allowed to grow, the permeability barrier is repaired in about two-thirds of a generation (129). The addition of chloramphenicol or tetracycline or the omission of required amino acids does not affect the repair rate. A proton pump inhibitor such as 2,4-dinitrophenol, however, prevents repair. The activity of omeprazole and lansoprazole against H. pylori (179) may be related to their inhibitory effect on certain cellular membrane pumps. Omitting glucose also prevents biofilm repair. Interestingly, the addition of macrolides decreases the repair rate, possibly because of its inhibition of mRNA translation (180) as well as by the elaboration of exotoxins by means of codon–anticodon interactions that inhibit the translation of mRNA for inducible enzymes (130–138).
Effects of Antimicrobial Agents on the Production of Biofilm
Antimicrobial agents, not unexpectedly, can either increase or decrease the production of biofilm. This effect, in part, appears species-specific. For example, fluoroquinolones at concentrations of one-half the MIC of Staphylococcus epidermidis decrease the production of slime (i.e., biofilm) (181). On the other hand, exposure of Klebsiella pneumoniae to a fluoroquinolone such as ciprofloxacin (182) has been shown to increase the amount of biofilm by more than 100-fold. Similarly, exposure of K. pneumoniae as well as many other microorganisms to β-lactam agents results in increased production of biofilm (182). Reduction of biofilm can be seen with other agents such as salicylates (183). The reduction of biofilm appears to occur concomitantly with a decrease in porin proteins (184–186). If these porins are being utilized to pump the biofilm precursors to the cell wall outer surface for final assembly, the two events are probably cause and effect. The decrease in biofilm and porin protein has been shown to result in resistance (185,187,188). This phenomenon has been shown to inhibit the activity of cephalosporins (188), aminoglycosides (183), and carbapenems (186). If biofilm is increased by exposure to β-lactam agents, then antimicrobial agents that have an effect on biofilm should be enhanced by preexposure of the bacteria to β-lactam agents. Indeed, this has been noted in both in vitro (189) and in vivo (190) studies.
Effects of Biofilm on Antimicrobial Action
The establishment of biofilm is an important aspect of cell wall physiology for individual cells and is equally important for microorganisms collectively. Bacteria that live and metabolize in these dense biofilm-encased microcolonies gain a number of the advantages enjoyed by multicellular life forms (52,56,57,191). One such advantage is a circulatory system (although primitive) with which to receive nutrients and into which to discharge metabolic wastes. This circulatory system consists of permeable channels that pass through less dense areas of biofilm interspersed within the dense microcolonies (56,191). Along these channels live river populations of microorganisms. These channels have convective flow patterns that permit the penetration and distribution into the biofilm matrix of large (2,000 Da) molecules. Dissolved oxygen is another critical commodity that is distributed within the biofilm through these channels. Microelectrodes have determined that the concentrations of dissolved oxygen in dense microcolonies approach zero at the center, owing to diffusion limitations and oxygen utilization (192). Such direct observations explain the need for anaerobic pathways for microorganisms such as P. aeruginosa (90) and M. tuberculosis (193) that are considered to be strict aerobes. Similar redox-sensitive chemical probes and autoradiography (194) have been used to detect metabolic activity and have demonstrated that, within a microcolony, the majority of cells are metabolically active. Although metabolically active, bacteria within biofilm colonies grow very slowly and are considered to resemble stationary-phase cultures (19,60). Moreover, those microorganisms downriver receive fewer substrates and may therefore become nutritionally starved, setting into motion the complex set of events (59,61) previously described.
Changes in microbial growth rate and nutrient limitation have long been recognized to cause changes in cell envelope components, which, in turn, influence the susceptibility of the microbes to antimicrobial agents (27,59,61,195). Establishment of biofilm is a growth-related factor that influences the susceptibility of the microbes to antimicrobial agents (196). For example, exposure of planktonic cells of P. aeruginosa to a biofilm surface produced by cells of the same species triggers the expression of at least two genes, algC and algG (74). This influences the susceptibility of these cells to antimicrobial agents, because sessile cells encased in biofilm are phenotypically different from planktonic cells of the same species (57,58,197).
The presence of biofilm at the individual cellular level contributes to changes in the overall susceptibility patterns of microorganisms involved in chronic infections, because the encasement by biofilm allows aggregates of cells to exist together in microcolonies. In mature biofilms, bacterial cells occupy only 5% to 35% of the biofilm mass (49,58,63,64,197). There are currently two leading hypotheses for the persistence of chronic biofilm-associated infections: (a) decreased concentrations of antibiotics caused by impaired transport to some regions of the biofilm (49,191,198,199) or by a dilutional effect (49) and (b) physiologic differences of sessile cells (197). A biofilm accumulation model has predicted that both mechanisms would result in reduced antimicrobial susceptibilities of 7-day-old biofilms compared with those of 2-day-old films (49). Growth rate–dependent killing was predicted to be decreased in thicker biofilms because of oxygen depletion, leading to reduced growth rates. The model also predicted resistance to the antibiotic due to depletion caused by increased biomass. The explanation was not that the antibiotic would fail to penetrate the biofilm but instead that the drug would be diluted in the bulk fluid. The binding of agents to biofilm is related to two factors: the relative availability of drug and the relative proximity. Relative availability is proportional to the amount of drug, whereas relative proximity is proportional to the concentration of the drug. The total amount of drug may remain the same as the biomass of polysaccharide increases, but the relative proximity decreases. Finally, many of the factors affecting antimicrobial susceptibility may change over time as the biofilm colony matures, because maturation alters the milieu for many microorganisms within the microcolony. For example, colonies deep within thick, mature biofilm may have reached the starvation stage.
Mechanisms of Antimicrobial Uptake
A factor that is clearly of great importance for effective antimicrobial action against bacteria is the penetration of the antimicrobial agent into both the human cell (200) and the microbial cell (201). Entry of the antimicrobial agents into human cells is considered a part of the pharmacokinetics of these agents (6). In this section, the entry of the antimicrobial agent into the microbial cell will be reviewed. In order to understand antimicrobial uptake, it is useful to understand the physiology of transport mechanisms located on microbial cell membranes.
In all microorganisms, the cytoplasmic membrane provides an osmotic barrier that is permeable to very few substances. It is porous to water and to uncharged organic molecules up to the size of glycerol. Gram-negative bacteria have an additional cell membrane, the outer membrane, which also acts as an effective barrier against antibiotics (155–157). In particular, hydrophobic antibiotics diffuse relatively poorly through the outer cell membrane in gram-negative bacteria in comparison with diffusion through the cytoplasmic membrane. This is due to the lack of glycerophospholipid in the outer portion of the cell bilayer, which instead consists largely of lipopolysaccharides.
Antimicrobial agents derived from microorganisms bear little resemblance to natural substrates brought into the bacterial cell but instead are more akin to metabolites excreted by cells (202). Therefore, with few exceptions (e.g., fosfomycin, which uses a stereospecific nutrient transport system) (203), antibiotics do not utilize active transport mechanisms for substrate uptake into bacteria.
There are three general mechanisms for substrate uptake into the bacterial cell: simple diffusion, facilitated diffusion, and active transport (158,204). There is a fourth mechanism known as the self-promoted uptake pathway, which is used by certain bacteria for the uptake of polycationic antibiotics (205). Each is discussed.
Simple or passive diffusion is defined as movement of molecules across a permeable membrane in which the flux in either direction is proportional to the concentration on the entering side and the rate of net transfer is proportional to the concentration differences between the two sides. An important factor in this type of diffusion is the partitioning coefficient, which essentially indicates the ability of the substrate to dissolve into the membrane interior (i.e., permeability). Simple diffusion kinetics occurs with nonpolar organic molecules such as tetracycline, which penetrates by dissolving in the lipid of the membrane, as well as with antimicrobial agents that move across a membrane through water-filled membrane-protein channel (i.e., pore) that is known as a porin (201,205–208). Fluoroquinolones, for example, are taken into bacteria by passive diffusion through porins in a passive diffusion process that exhibits nonsaturable kinetics. Fluoroquinolones are amphoteric molecules and have both zwitterionic and uncharged forms at neutral pH. Generally, only uncharged molecules are involved in the passive diffusion process, with the amount of uncharged forms greatly influencing the penetrating ability of these agents. Charged molecules can also exhibit passive diffusion, provided there is an electrical gradient across the membrane (209).
Facilitated diffusion in theory involves a barrier-insoluble substance reacting with a carrier (i.e., transporter) within the barrier to form a complex that can shuttle across the membrane, where the substance is then released. This type of diffusion exhibits saturable Michaelis-Menten kinetics with a Km and a Vmax, but the Km is the same on both sides of the membrane because this mechanism is not linked to an energy source. Another name for this type of uptake pathway is a passive carrier–mediated system. Facilitated diffusion is seen in yeasts (210) but has not yet been identified in bacteria.
Active transport means that the bacterial cell has the ability to concentrate molecules within the cell. This ability can be turned on or off (i.e., is inducible) and is linked to an energy source. Without this energy source, the molecules cannot pass across the membrane. The kinetics of active transport exhibits a Km and a Vmax, like the activity of an enzyme, and the carrier system can be saturated.
The self-promoted uptake pathway has been described for gram-negative bacteria and involves binding of the antibiotic to the lipopolysaccharide in the outer membrane (205,211,212). This is followed by outer displacement, by the antimicrobial agent, of magnesium and possibly calcium ions in the lipopolysaccharide matrix of the biofilm (213). This causes instability of the biofilm matrix, as described earlier, and leads to increased permeability (214). The presence of additional divalent cations prevents this by stabilizing the complex. The self-promoted pathway was first described as an uptake mechanism in P. aeruginosa for polycationic antibiotics such as polymyxin and the aminoglycosides (213). More recently, it has been noted for azithromycin in E. coli (215).
Cell membranes of microorganisms must be energized in order to concentrate nutrients needed for growth. The electrochemical gradient–induced proton motive force is a key factor in these energized cytoplasmic membranes. Microbial cell membranes are intrinsically impermeable to protons yet must move protons in or out of the cell. For example, any change in the intracellular pH of the microorganism would need to be absorbed by the buffering capacity of the cytoplasm (148) unless there was some method for expelling protons. Such a pH-homeostatic method exists and involves membrane-bound proton pumps (149). Proton-driven translocation of molecules is facilitated by reduced pH (216). These pumps may at times be responsible for the efflux of antibiotics by pumping out protons that are complexed with a negatively charged antibiotic.
The outer membrane in gram-negative bacteria, through changes in porin diffusion channels, can serve as a permeation barrier and thus greatly influences antimicrobial resistance (156,157,178,217,218). Moreover, the uptake of antimicrobial agents into bacterial cells also can be influenced by efflux mechanisms that may concomitantly act to remove the agent (150,156,219–222). In fact, the antimicrobial activity may be determined by the race between uptake and efflux. It is useful to appreciate the mechanisms of efflux, because these should themselves prove to be excellent targets for antimicrobial agents (222–225).
Membrane-bound proton pumps may be readily overcome by compounds with uncoupling activity (219). Classic uncouplers include carbonyl cyanide-m-chlorophenylhydrazine and 2,4-dinitrophenol (226). These uncouplers result in the abolition of respiratory control in the bacteria. This, in turn, results in stimulation of respiratory activity.
INHIBITORY OR LETHAL ANTIMICROBIAL ACTIVITY VERSUS FUNCTIONAL SYNERGY AGAINST MICROBES
Inhibitory and Lethal Effects
The goal of antimicrobial therapy, as appreciated by Lister and Ehrlich, is to destroy the invading microorganism without harming the host. The effectiveness of an antimicrobial agent has traditionally been measured by its ability to inhibit and kill bacteria. In theory, there are three basic ways to kill a bacterial cell: by causing irreparable damage to its genome, to its envelope, and to certain classes of its proteins (114–121). Antimicrobial agents have been developed that attempt to kill bacteria in each of these ways. Often, several antibiotics that use two of these three different ways are combined to enhance the lethal effect. Yet, as already noted, bacteria are not particularly easy to kill. This fact has not escaped microbiologists. It is well known that most antimicrobial agents exert their lethal effects on bacteria during the growth phase (2,27,195). Therefore, microbiologists have designed routine susceptibility tests to measure antimicrobial activity during the logarithmic growth phase in media that provide all of the ingredients for optimal growth (62). However, this is not the usual state of microorganisms in infected tissues. For example, S. aureus isolates from tissue-cage infections in rats have been shown to be in a state of dormancy and thus are relatively resistant to most antimicrobial agents (227). Perhaps the closest that broth susceptibility testing comes to mimicking a clinical infection is in the case of acute bacterial meningitis. Even then, there clearly is room for improvement (228). Susceptibility testing must be repositioned to provide test results that correlate with the clinical infection. Fortunately, clinical microbiologists have been working to accomplish this goal. An example is the integrated use of pharmacokinetic and pharmacodynamic models for the definition of breakpoints (9). As a result, the correlation of in vitro susceptibility testing with in vivo clinical effectiveness has markedly improved (14). Moreover, clinical microbiology laboratories are aware of emerging mechanisms of resistance and are particularly vigilant in detecting such resistance (229,230). Finally, antimicrobial susceptibility testing is frequently integrated with an antimicrobial stewardship program (231) that is aimed at controlling resistance.
Most clinical infections involve bacteria in a sessile state, as opposed to a planktonic state (63). However, antimicrobial agents that are able to kill bacteria in their sessile state are few in number (28,232,233). Of those agents currently available for clinical use, carbapenems and the fluoroquinolones have the greatest lethal effect against sessile bacteria—a lethal effect more readily obtained against gram-negative isolates than against gram-positive ones (233–235). Against gram-positive pathogens such as staphylococci, daptomycin has the greatest lethal effect against stationary-phase and nondividing organisms (236,237). This in vitro bactericidal activity, often defined as equal to or greater than a 3 log10 decrease in colony-forming units over a 24-hour period (62), may not be sufficient for total microbial killing against certain microorganisms (48) or with certain infections such as endocarditis (238). Total and rapid microbial killing is important in acute bacterial meningitis as well as in acute sepsis in immunodeficient hosts (2,7). Even the most rigorous in vitro susceptibility test methods, including time-kill kinetic methodology, may provide misinformation if the growth phase of the microorganism and the clinically desired end point are not correlated with the test method (14). This is shown by a report by investigators who found that clarithromycin, like other macrolides, demonstrated in vitro bactericidal activity against pneumococci by time-kill kinetic methodology (239). However, in a rabbit model for pneumococcal meningitis, clarithromycin was unable to cure pneumococcal meningitis despite susceptible isolates and cerebrospinal fluid levels of clarithromycin comparable to those used to achieve in vitro killing (239). In chronic infections such as endocarditis and osteomyelitis, where involvement of biofilm is almost always present, rapid microbial killing is usually not achievable due to factors such as decreased biofilmpenetration/dilutional effects and dormant growth phase (240,241). Total microbial killing, on the other hand, is a well-recognized goal when treating these particular infections; antimicrobial therapy is usually given for 6 to 8 weeks in order to achieve total microbial killing (242,243).
The use of bactericidal drugs in order to achieve total microbial killing is not required for most infections (244,245). However, when bacterial eradication is deemed necessary, there are a number of ways that this may be predicted (246). Pharmacokinetic and pharmacodynamic parameters may be useful for predicting bacterial response (246,247). In addition, there are bactericidal tests including proposed reference methods available in most clinical microbiology laboratories (248).
The accumulated knowledge of antimicrobial mechanisms of action on different growth phases of bacteria has reached a point where it may encourage a multicomponent drug approach to antimicrobial therapy (21,249,250). This approach may involve using combination therapy that achieves functional synergy against the infecting microorganism. Functional synergy directed against the microorganism often can be achieved by using knowledge of the various physiologic states within which microorganisms exist combined with knowledge of specific antimicrobial agents that interfere with each of the physiologic states. When these agents are combined, their use may result in enhanced microbial killing that can be thought of as functional synergy. Although enhanced killing, as defined by a strict definition of synergy, may not be detected using in vitro methods, enhancement of total killing may be measured in other ways. For example, animal models have long been used to assess the ability of antimicrobial agents alone or in combination to eradicate microorganisms (251).
Functional synergy as a therapeutic approach is already in use but is not well appreciated. The therapy of TB is one of the oldest examples of this approach, for in such therapy, multiple antituberculous agents result in enhanced mycobacterial killing as well as minimizing the emergence of resistance. An example of this therapeutic approach is the recognition of the lethal effect of metronidazole on dormant forms of M. tuberculosis (252). This lethal activity appears to be related to the fact that the dormant state requires anaerobic pathways, which then provide the necessary electrons to activate metronidazole to its electrophilic degradation products (252). Metronidazole alone does not reduce the bacillary burden of M. tuberculosis in the guinea pig model (253). Exposing M. tuberculosis in its dormant state to metronidazole apparently kills the organism or triggers aerobic respiration. The return to aerobic pathways does not occur independently but instead occurs with resumption of mycobacterial replication. Thus, combining metronidazole or similar agents with agents that interfere with the replicating stage creates functional synergy that allows enhanced killing of the mycobacterium (24,254). Drug development strategies that target the different physiologic states of M. tuberculosis including the latent phase may allow improved treatment of TB (255). Indeed, the addition of metronidazole to isoniazid (INH) and rifampin in the macaque model has been shown to effectively treat animals with active TB within 2 months (256).
Another example of functional synergy is the use of an aminoglycoside with a β-lactam agent for the therapy of infections such as bacterial endocarditis caused by P. aeruginosa. The increased effectiveness of this combination is due to the disruption of the biofilm by the aminoglycoside (173,257), which enhances the β-lactam agent in two ways. The first is when disruption provides holes in the bacterial cell wall that allow increased penetration of the β-lactam agent. The second is when the dormant form is forced to replicate in an attempt to fix the damaged cell wall, thus providing a target for the β-lactam agent.
Other chronic infections that have benefited from a multidrug approach that provides functional synergy include pulmonary infections caused by P. aeruginosa in patients with cystic fibrosis. These infections involve the establishment in lung tissue of biofilm-encased microcolonies in which are found some dwarf forms, which may represent dormant forms that are utilizing anaerobic pathways (90,104,105). Clinical cure of these Pseudomonaspulmonary infections is rarely achieved (104). This is consistent with the in vitro observation that total microbial killing of sessile strains of P. aeruginosa is extremely difficult to achieve after the biofilm has matured for 5 to 7 days (196). Neither older synergistic combinations such as tobramycin and piperacillin (77) nor newer synergistic combinations such as fosfomycin and ofloxacin (258) are able to achieve total killing.
There are, however, some approaches that may allow functional synergy. The use of aerosolized tobramycin (259,260) or aerosolized colistin (261) is one of these; this approach provides greater concentrations of a biofilm disrupter (i.e., both tobramycin and colistin) as well as a bacterial cell membrane disrupter (i.e., colistin), which then can enhance the systemic use of other antipseudomonal agents. The prolonged use of aminoglycosides in chronic Pseudomonas infections is known to be followed by the emergence of aminoglycoside-resistant Pseudomonas strains characterized by a deep rough colony morphology on agar plates due to the lack of biofilm (257). The lack of a lipopolysaccharide/biofilm target for the primary action of the aminoglycoside is the mechanism of resistance, because these strains do not exhibit altered ribosomes or produce aminoglycoside-inactivating enzymes. The outer cell walls of these aminoglycoside-resistant strains are characterized by the lack of lipopolysaccharide and a marked increase in the amount of OprH OMP (257). Overproduction of this outer cell membrane protein decreases the accumulation of polymyxin and gentamicin by the self-promoted pathway (214). However, overproduction of OprH is associated with increased susceptibility to fluoroquinolone antibiotics (262). It appears that the overproduction of OprH is a mechanism that minimizes biofilm in order to counter the effects of biofilm disruption by polycationic agents, but in doing so, the altered cell wall seems to offer increased diffusion of lipophilic fluoroquinolones into the cytoplasm. The use of a fluoroquinolone and an antipseudomonal β-lactam agent along with the aerosolized aminoglycoside (264) thus allows functional synergy and provides an additional therapeutic option (265). Moreover, the addition of a macrolide such as azithromycin or clarithromycin to this regimen may prove useful. These macrolides have been shown to decrease the production of both biofilm (135,137,266) and exoenzymes (131,132) by P. aeruginosa, which creates yet another biofilm-related conflict while preventing further pulmonary damage by the exoenzymes (132,266–271). Macrolides and ketolides have been shown to have an effect on the outer membrane of P. aeruginosa (272,273) that appears to potentiate the activity of antipseudomonal agents and may allow functional synergy (274). Clinical experience with the use of aerosolized tobramycin (259,260), fluoroquinolones (264), and macrolides (275) for exacerbations of Pseudomonas infections in cystic fibrosis patients have shown that multidrug regimens that include one or more of these three agents result in reductions in P. aeruginosa sputum density and improvements in pulmonary function (265). Interestingly, a similar approach using a combination of clarithromycin and ceftazidime in a rat model for foreign body–related osteomyelitis caused by P. aeruginosa demonstrated that clarithromycin eradicated the biofilm and enhanced the bactericidal effect of ceftazidime (276).
There are other examples of functional synergy in microbes that can be purposely created by the selective use of antibiotics. Enhanced microbial killing is a frequent goal of combination antimicrobial therapy for infective endocarditis (20). Therefore, a number of examples of functional synergy demonstrated in experimental endocarditis are discussed. Temafloxacin has been shown to be effective in the therapy of experimental streptococcal endocarditis, and studies have found it to penetrate vegetations in a homogeneous manner (277). Dextranase is an enzyme capable of hydrolyzing 20% to 90% of streptococcal glycocalyx (biofilm). When used alone, dextranase has no in vitro antimicrobial effect on viridans streptococci nor does it have a beneficial effect on experimental streptococcal endocarditis (278). When dextranase is used in combination with temafloxacin, it significantly potentiates the effect of temafloxacin in vivo by reducing the amount of bacterial biofilm in infected vegetations and by altering the metabolic status of the microorganisms (279). The same effect has been seen when dextranase and penicillin have been combined in the treatment of experimental streptococcal endocarditis (278). Finally, an animal model for experimental P. aeruginosa endocarditis has shown an identical effect for alginase combined with amikacin (280). Of importance is the lack of beneficial effect demonstrated when the vegetation size is reduced by fibrinolytic therapy alone (281,282). The results of these studies are consistent with the theory that the bacteria in microcolonies embedded in biofilm have a lower metabolic rate (283). Reducing the amount of biofilm results in both an increased metabolic rate and increased replication, which each increases the antimicrobial activity of most antibiotics.
Another experimental approach to disrupting the biofilm as a method for creating functional synergy is to use the proteolytic enzyme serratiopeptidase (284). Serratiopeptidase is a metalloprotease produced by a strain of Serratiathat is only partially inhibited by in vivo protease inhibitors and has been used as an antiinflammatory drug because of its ability to increase the penetration of antibiotics into infected sites (285). This protease has been found to enhance the activity of ofloxacin on sessile cultures of P. aeruginosa and S. aureus (286). In addition, serratiopeptidase has been shown to reduce the ability of Listeria monocytogenes to form biofilm and to invade host cells (287).
Another commonly used drug with the potential for creating biofilm-related functional synergy in the therapy of microbial infections is aspirin (288). Aspirin has been noted to be a cell wall permeabilizer for P. aeruginosa (178). This role as a cell wall permeabilizer may be related to its effect on biofilm. Aspirin has been shown to cause a dose-dependent reduction in the weight of aortic vegetations in experimental endocarditis (289). In addition, when combined with vancomycin, aspirin improves the sterilization rate of aortic valve vegetations infected with S. aureus. These effects are similar to those of dextranase (279) and the protease of Serratia (286) and may be a result of functional synergy. Aspirin has been found to diminish the amount of microbial biofilm in a number of other studies (183,288). Salicylates also are known to depress the synthesis of porins in E. coli, K. pneumoniae, Serratia marcescens, Burkholderia cepacia, and P. aeruginosa (184–186,188). If these depressed porins are involved in the efflux of biofilm precursors as a part of biofilm maintenance, these two physiologic phenomena may be related. Finally, the combination of aspirin and amphotericin B has demonstrated functional synergy against biofilm cells of Candida albicans and Candida parapsilosis versus indifferent effects against planktonic cells of these microorganisms (290). Each of these antimicrobial strategies against infectious bacterial biofilm (291) is an example of the use of functional synergy.
Finally, rifampin combination therapy is a very controversial multidrug approach to a number of nonmycobacterial infections (292,293) that may owe its somewhat surprising albeit unpredictable efficacy to functional synergy. Rifampin combination therapy has been used clinically for various types of infections (293), but the predominant use seems to be for staphylococcal infections (292) that involve osteomyelitis and/or a prosthetic device–related infection. The controversy stems from a lack of convincing in vitro data that support the in vivo clinical findings. There are a number of clinical studies involving bone or joint infections that have demonstrated such in vivo efficacy, although these studies are generally underpowered (294–301). Examples of a number of these clinical studies are provided. In one study (296), therapy with 900 mg/day rifampin plus 600 mg/day ofloxacin for 6 months was used for patients with prosthetic implants infected with Staphylococcus spp. The overall success rate was 74% among 47 patients, with 62% of patients being cured without removal of their orthopedic device (296). The success rate was 81% for the hip prosthesis group, 69% for the knee prosthesis group, and 69% for the osteosynthesis device group. A total of eight treatment failures were related to the isolation of a resistant microorganism. In another study (297), 33 patients with culture-proven staphylococcal infection associated with stable orthopedic implants and with a short duration of symptoms of infection were treated: 18 patients received ciprofloxacin and rifampin, whereas 15 patients received ciprofloxacin and a placebo. Twenty-four patients completed the trial; the cure rate was 12 of 12 (100%) for those who received ciprofloxacin plus rifampin versus 7 of 12 (58%) for those who received ciprofloxacin plus a placebo (297). In a third study (295), 10 patients with Staphylococcus spp–infected orthopedic implants were treated with various antibiotic regimens, all of which included rifampin. Of these patients, 8 were cured. Many of these studies (296–298,301) combined rifampin with a quinolone; indeed, early results with such oral therapy using rifampin combined with a quinolone have been encouraging (302).
Animal models of adjunctive rifampin for therapy of Staphylococcus-infected prosthetic devices/foreign bodies/osteomyelitis may offer some additional insight. A rat model of chronic staphylococcal foreign body infection (227) demonstrated that antimicrobial combinations of fleroxacin plus vancomycin and vancomycin plus fleroxacin and rifampin were highly effective and superior to single drugs. Further, the three-drug therapeutic regimen decreased bacterial counts more rapidly than the two-drug therapy and was curative in most cases (92% for three drugs versus 41% for two and less than 6% for monotherapy). No mutants resistant to these three agents were detected with combination therapy. A rabbit model of acute staphylococcal osteomyelitis caused by MRSA noted a 100% infection clearance with tigecycline and rifampin versus a 90% clearance with tigecycline alone, whereas vancomycin and rifampin showed a 90% infection clearance versus an 81.8% clearance with vancomycin alone (303). A guinea pig model assessing linezolid alone or combined with rifampin against a foreign body infection caused by MRSA demonstrated that the efficacy in the eradication of cage-associated MRSA infection was achieved only with the combination of rifampin and linezolid, with cure rates being between 50% and 60%; in this guinea pig model, a levofloxacin–rifampin combination achieved a 91% cure rate against a quinolone-susceptible MRSA strain (304). An MRSA knee prosthesis infection in rabbits was used to evaluate daptomycin or vancomycin alone and in combination with rifampin (305). This study demonstrated that daptomycin combined with rifampin sterilized 11 of 11 bones versus 2 of 12 for daptomycin alone, whereas vancomycin combined with rifampin sterilized 6 of 8 bones versus 0 of 12 for vancomycin alone (305). Moreover, rifampin prevented the emergence of daptomycin-resistant MRSA; the authors concluded that adjunctive rifampin is crucial to optimizing daptomycin efficacy against rabbit prosthetic joint infection due to MRSA (305). Additional studies in animal models have demonstrated similar efficacy of rifampin combinations (306–308).
The success of adjunctive rifampin for the therapy of Staphylococcus-infected prosthetic devices/foreign bodies/osteomyelitis may once again be due to functional synergy that targets biofilm (241,291). The efficacy of fluoroquinolones (302) is of interest because fluoroquinolones have been shown to decrease the production of slime (biofilm) by S. epidermidis (181), and it is likely that rifampin, through inhibition of protein synthesis, may decrease or prevent the availability of critical enzymes needed for ongoing maintenance of biofilm. As the biofilm microcolonies attached to the prosthetic device or glued into the bone begin to be slowly disrupted by the lack of ongoing maintenance, the staphylococci are forced to replicate, which further enhances the antimicrobial action of each antibiotic.
MECHANISMS OF ACTION FOR SELECTED CLASSES OF ANTIMICROBIAL AGENTS
Antimicrobial Classes in Current Clinical Use
As previously described, the main structural features of the peptidoglycan sacculus are linear glycan chains interlinked by short peptide bridges. A number of enzymatic activities are involved in the biosynthesis of the sacculus: catalyzation by glycosyltransferase enzymes of the formation of the linear glycan chains, cross-linking of the glycan chains by transglycosylase enzymes, and cross-linking via peptide bridges by transpeptidase enzymes (32,151). The latter peptide cross-links provide mechanical strength against osmotic pressure forces. Peptidoglycan structural modifications of completed cell wall are required in replicating cells as they grow, and each microorganism therefore possesses specific peptidoglycan hydrolases that are responsible for such structural adjustments (38–40,309). It is these transpeptidases/hydrolases (39,40,309), as well as other factors such as activation of newly recognized apoptotic death pathways (42–45), that appear to be important target(s) of β-lactam agents (36,40,96,310–314).
The mechanism of action of β-lactam agents is more complex than initially thought and likely involves three interrelated cellular processes (314). The first of these cellular processes is transpeptidation, which initially was thought to be the sole target of β-lactam agents (310). Penicillins, because of their structural similarity to the C-terminal D-alanyl-D-alanine end of the peptide stem, react chemically with the transpeptidases, also known as PBPs, to form stable acyl-enzymes, inactivating the PBPs and preventing further cross-linking. The inhibition of glycan cross-linking then leads to a weakened cell wall, which eventually ruptures due to osmotic pressure. However, it was noted that penicillins were able to cause inhibition of growth in certain bacteria without bacteriolysis. Therefore, triggering of autolytic cell wall enzymes was considered as a second and separate target of β-lactam agents (43,127,311). However, the mechanism for control of the autolytic system and how it was activated during treatment with β-lactam agents remained unknown until a number of observations suggested several possibilities. The electrophysiologic state of the cellular membrane is thought to be an important factor in the regulation of bacterial cell wall autolysis (107,315,316). There is increasing evidence that β-lactam agents may depolarize the membrane potential as a signal to induce autolysis (44,45,317). Moreover, there are a number of regulatory genes that are involved in bacterial autolysis (97,318,319). These genes may be activated after exposure to a sufficient concentration of β-lactam agent to cause irreparable damage (30,95,319). For example, a signal transduction pathway involved in regulating apoptotic death in pneumococci has been described (96). One of the death signals appears to be a peptide, which may function in a quorum-sensing manner. Finally, metabolism-related depletion of NADH, leaching of iron from iron-sulfur clusters, and stimulation of the Fenton reaction has been shown to lead to formation of harmful hydroxyl radicals that triggers an oxidative damage cellular death pathway (44,45). This pathway may be modulated by the stringent response in a manner yet to be detailed. Modulation of the stringent response under antimicrobial selection appears to create mutants that are virulent and not killed by a broad spectrum of antimicrobial agents (55). This resistance phenomenon has been described as physiologic tolerance (28,320).
Penicillins are characterized by a four-membered β-lactam ring fused to a five-membered thiazolidine ring containing a side chain (321). Manipulations of this side chain have been important in the pharmacokinetics and pharmacodynamics of penicillins. The ability to produce 6-aminopenicillanic acid (6-APA) by fermentation allowed chemists to replace the amino group of 6-APA with a large number of altered side chains, thus producing many different semisynthetic penicillins (322). The steric hindrance around the amide bond produced by bulky side chains such as carbocyclic or heterocyclic rings with substituents at the orthoposition of the 6-APA site produced the first semisynthetic penicillins with increased stability against staphylococcal β-lactamase. A number of such antistaphylococcal penicillins with bulky side chains have been synthesized, including methicillin; nafcillin; and the isoxazolyl penicillins, oxacillin, cloxacillin, dicloxacillin, and flucloxacillin.
Semisynthetic penicillins also include those created by a simple replacement of the α-carbon of the hydrophobic side chain at position 6 of benzylpenicillin by an amino (e.g., ampicillin), a carboxyl (e.g., carbenicillin), a ureido (e.g., mezlocillin), a piperazino (e.g., piperacillin) group, or a methoxy (e.g., temocillin). The result was the development of the extended-spectrum penicillins, which have been grouped as aminopenicillins, carboxypenicillins, ureidopenicillins, and methoxypenicillins (322–325). Such substitutions provided improved penetration through the outer cell membrane of gram-negative microorganisms as well as increased stability against β-lactamases produced by these pathogens. In particular, penetration through the restrictive pores of P. aeruginosa resulted in antipseudomonal activity for the carboxypenicillins (e.g., carbenicillin and ticarcillin) and the ureidopenicillins (e.g., mezlocillin, azlocillin, and piperacillin). The only methoxypenicillin in clinical use is temocillin (324,325), which is not active against gram-positive organisms, anaerobes, and Pseudomonas species; temocillin has not been approved for use in the United States. Temocillin is, however, resistant to most if not all classic and extended-spectrum β-lactamases as well as to AmpC β-lactamases (324). For this reason, it has been used in England as a carbapenem-sparing agent (324,325). Because the stability against β-lactamases did not include staphylococcal β-lactamase, a number of these extended-spectrum penicillins were combined with a β-lactamase inhibitor (e.g., clavulanate, sulbactam, or tazobactam) and are known as β-lactam–β-lactamase inhibitor combinations (323,326,327). To date, these combinations include amoxicillin/clavulanate, ampicillin/sulbactam, ticarcillin/clavulanate, and piperacillin/tazobactam.
Cephalosporins are characterized by a four-membered β-lactam ring fused to a sulfur-containing ring-expanded system (328). One of the first cephalosporins, cephalosporin C, possessed an aminoadipic side chain, which could easily be chemically removed to give rise to 7-aminocephalosporonic acid (7-ACA), which is analogous to 6-APA (329). From 7-ACA came the first-generation semisynthetic cephalosporins such as cefazolin. Substitutions at the C7 position as well as at the C3 position of the dihydrothiazine ring allow greater variation of semisynthetic cephalosporins than can be achieved with the penicillins (322,323). Consequently, more cephalosporins have been developed, and detailed reviews of these agents are available (322,323,328,330). Side chain substitution in the cephalosporins is built on the experience with penicillins and includes thiazolyl and phenylglycyl side chains. The substitutions at the C7 position are of particular importance in governing stability against β-lactamases. For example, substitution at the C7-α position of cephalosporins with a methoxy group (e.g., cefoxitin and cefotetan, which are second-generation cephalosporins) resulted in the cephamycins, which have increased stability against β-lactamases, including those of the Bacteroides fragilis group. Substitution at the C7-β position with a methoxyimino group (e.g., cefotaxime, which is a third-generation cephalosporin) also increased the resistance of these agents to β-lactamases. Acyl side chains used with cephalosporins include aminothiazole oximes, which may have charged carboxylates (e.g., ceftazidime, which is a third-generation cephalosporin) that improve penetration through gram-negative bacterial outer membranes. Cefepime, a fourth-generation cephalosporin, also has a positively charged quaternary ammonium in position C3, which creates a zwitterion that allows increased penetration of the gram-negative bacterial outer membrane. The 2-aminothiazolylacetamido group found in cefepime provides increased stability against β-lactamases. Ceftaroline is the first member of a new subclass of β-lactam agents, cephalosporins with activity against MRSA (331). A 1,3-thiazole ring attached to the 3-position of the cephalosporin nucleus and the oxime group in the C7 acyl moiety provide the basis for increased activity against MRSA with the 1,3-thiazole ring binding tightly to the MRSA PBP 2A following a conformational change in the protein allowing the active site to be exposed for binding (332). Ceftaroline is also active against multidrug-resistant S. pneumoniae.
Carbapenems differ from conventional penicillins in having no sulfur atom in their five-membered ring and in having a double bond between carbons 2 and 3 (332,333). This sterically alters the cis/trans configuration of the molecule in comparison with other β-lactam agents and places the amide bond away from the water-containing groove of the serine-based β-lactamases. However, carbapenems are susceptible to hydrolysis by metallo-β-lactamases. Four carbapenems (imipenem, meropenem, ertapenem, and doripenem) are approved for clinical use in the United States.
There are two monocyclic β-lactams produced by microorganisms that possess antimicrobial activity, nocardicins and monobactams (334). The monobactam nucleus of these compounds exhibits only weak antimicrobial activity, and they, like the penicillins and cephalosporins, must have substitution around the central nucleus to achieve clinically useful antimicrobial activity. Side chain structure–activity relationships in monobactams parallel those of penicillins and cephalosporins. There is only one monobactam antibiotic, aztreonam, that has a 3-acyl aminothiazole-oxime side chain identical to that of ceftazidime, while the lactam ring has a N-sulfonate substituent on the other side (330,332,334). The N-sulfonate substituent is essential for β-lactamase stability. Like ceftazidime, aztreonam is useful only against gram-negative pathogens, with good activity against P. aeruginosa.
All aminoglycoside antibiotics contain one or more amino sugar residues linked to a central, six-membered aminocyclitol ring by pseudoglycosidic bond(s). Spectinomycin is, strictly speaking, also an aminocyclitol with three fused rings but lacks amino sugars and pseudoglycosidic bonds. The primary mechanism of action of aminoglycosides is a decrease in protein synthesis after the drug has bound to the prokaryotic ribosome at the 16S ribosomal RNA (rRNA) site located in the small (30S) subunit of the ribosome (335–339). Aminoglycosides are hydrophilic sugars with multiple amino groups that function as polycations. Their polycationic nature allows binding to the polyanionic 16S rRNA on the 30S ribosome at the A site for aminoacyl–tRNA binding (338,339). The A site is composed of portions of the 530 loop, helix 34, and the base of helix 44; transfer RNA (tRNA) anticodons bind in a cleft formed between these individual domains of the A site. Binding of the aminoglycoside to the A site inhibits the translation process by causing misreading and/or hindering the translocation step. Analysis by X-ray crystallography suggests that the polycation binds to the RNA bases rather than to backbone atoms. Moreover, attachment at the A site suggests that aminoglycosides block a required transformational transition during the peptide bond–forming translocation process, bringing the translocation steps of protein synthesis in the ribosome to a halt. Aminoglycosides also bind to helix 69 of the large (50S) subunit of the ribosome (340,341). The result of this binding is reduction in the mobility of an adenine residue at position 1492 of the rRNA A-site; this reduction in mobility may be a key determinant in the antibacterial activity of aminoglycosides (342).
The polycationic nature of aminoglycosides also accounts for their recognized effect on biofilm and cell membranes (163,172). Aminoglycosides are bactericidal agents and often exhibit a rapid lethal effect on susceptible aerobic gram-negative bacilli. Such a rapid lethal effect has been noted to be contrary to the expected effect of agents acting on ribosomal targets (173). This lethal effect of aminoglycosides against aerobic gram-negative bacilli, moreover, is concentration-dependent, with increasing concentrations achieving increased killing. Their effect against gram-positive cocci is, at best, inhibitory, unless a β-lactam agent is used in combination with the aminoglycoside.
Inhibition of protein synthesis, however, usually does not produce a bactericidal effect, let alone a rapid one. Therefore, binding to the 30S ribosome may not be the only mechanism of antimicrobial action for aminoglycosides; in fact, many susceptible gram-negative bacilli may be dead long before the drug arrives at the 30S ribosome (173,174,212). It is now recognized that aminoglycosides are polycations that competitively displace cell biofilm–associated Mg2+ and Ca2+ linking the polysaccharides of adjacent lipopolysaccharide molecules (174,211–213). The result is shedding of cell membrane blebs, with formation of transient holes in the cell wall and disruption of the normal permeability of the cell wall (163,170,173,174). This action alone may be sufficient to kill many susceptible gram-negative bacteria before the aminoglycoside has a chance to reach the 30S ribosome (Fig. 10.3). The surface effect of gentamicin has been investigated using bovine serum albumin–gentamicin complexes (174), which have been shown to be bactericidal against P. aeruginosa. These findings are in agreement with similar studies done with other immobilized surface agents (146,343–345).
Increased understanding of the mechanisms of action of aminoglycosides brought with it the realization that aminoglycosides have a concentration-dependent bactericidal effect (346) as well as a considerable postantibiotic effect (2). This allowed the dosing schedule to be modified to once per day (347). The modification integrated both pharmacokinetic and pharmacodynamic properties and offered the potential for greater efficacy and less toxicity (8,347–350). There has now been considerable experience with once-daily aminoglycoside regimens (347–350). Such regimens indeed appear to be clinically effective while reducing the incidence of nephrotoxicity. Moreover, they reduce the cost by decreasing ancillary service time and the need for serum aminoglycoside determinations.
Another question about the optimal utilization of aminoglycosides can be addressed. This concerns the timing of the doses when both an aminoglycoside and a β-lactam agent are administered. In vitro (189) and in vivo (190) studies have clearly demonstrated a remarkable advantage gained from nonsimultaneous administration of aminoglycosides and β-lactam agents when they are used in combination. The main benefit is a marked delay in bacterial regrowth regardless of the order in which the agents are given. However, the initial bactericidal effect is greater when the aminoglycoside is given first. The minimum time interval between doses of the aminoglycoside and the β-lactam agent for maximum delay of regrowth is 2 hours.
The reason for this phenomenon is clear if one accepts the premise that the aminoglycoside has its primary antimicrobial effect on biofilm. This effect would be greatest when there was no prior or concomitant use of cell wall–active agents, which have been shown to markedly affect the bacterial cell surfaces (103) and would thus lessen the opportunity for the aminoglycoside to have the optimal biofilm targets.
The bactericidal effects of aminoglycosides combined with β-lactam agents in vitro have been shown to be dependent on the concentrations of β-lactam agents used. If the concentration of the β-lactam agent is not optimized for bactericidal activity (i.e., four- to eightfold higher than the MIC), the effect of the β-lactam on the test microorganism is to stimulate increased production of biofilm (182) and thus enhance the effect of the aminoglycoside by providing a better target. Lorian and Ernst (351) have demonstrated this concept nicely. If, on the other hand, the concentration of the β-lactam agent is high enough to disrupt the cell wall morphology (103), then the biofilm target is lessened, which in turn lessens the lethal effect of the aminoglycoside (174,196). These two effects are exactly the opposite of what would be predicted to occur if the target of the aminoglycoside was the ribosome rather than biofilm. The overall lethal effect of aminoglycosides in vivo is enhanced by administering the aminoglycoside before the β-lactam agent (190). Historically, aminoglycosides have been used in combination with other antimicrobial agents (most often a β-lactam agent) in order to enhance microbial killing and achieve increased efficacy (349). Most studies, however, fail to demonstrate improved outcomes (349); this may be due, in part, to not administering the aminoglycoside first.
Macrolides, Azolides, Lincosamides, Ketolides, and Streptogramins
The macrolides, azolides, lincosamides, ketolides, and streptogramins are grouped together despite structural differences because of similar biologic properties, including their mechanism of action against the 50S subunit of the bacterial ribosomes. Bacterial ribosomes are an important target for antimicrobial agents and have two specific subunits that are targeted, the 30S ribosomal subunit and the 50S ribosomal subunit (118). The specific target for the 50S ribosomal subunit for many of these agents appears to be domain V of the 23S rRNA, which is the peptidyltransferase center (352,353). Macrolides block the approach to the exit tunnel for elongating peptides and thus prevent polypeptide translation, causing premature release of peptidyl–tRNA intermediates. Lincosamides, however, inhibit the initiation of peptide chain formation (134,354), whereas the effect of the other macrolides is to prevent the extension of the growing peptide chain. Macrolides also block assembly of 50S subunits by their interaction with the 23S rRNA. There are interspecies variations in the ribosome structure that affect the binding of macrolides (355). These interspecies variations not only affect wild-type ribosomes but also affect ribosomes that have acquired resistance mutations. Thus, a mutation that results in high-level resistance to a specific macrolide in a particular species may only produce low-level resistance or none at all in a different species (355,356). Finally, it should be noted that the 23S rRNA of bacteria is the homolog of human mitochondrial 16S rRNA; antimicrobial agents that inhibit the bacterial 23S peptidyltransferase center may also impair human mitochondrial function (357).
Ketolides are the latest members of the macrolide group and are novel semisynthetic 14-membered–ring macrolides in which the main structural innovations are the lack of the neutral sugar cladinose in position C3 as well as a C11/C12 carbamate (358–360). When the C3 cladinose sugar moiety is removed, the resulting 3-hydroxy group is oxidized to a 3-keto group, hence the name ketolide. The macrolides, azolides, lincosamides, and ketolides all appear to bind to the same site or contiguous sites on the ribosome and so they may become competitive inhibitors if used together (359,361,362). This mechanism of action, inhibition of protein synthesis, results in bacteriostatic activity against most bacteria by all of these agents except ketolides, which have bactericidal activity (358–360). Inhibition of critical proteins in certain microbial species does, however, result in bactericidal activity of the macrolides. Such species-specific bactericidal activity is seen in vitro with the macrolide clarithromycin against S. pneumoniae but is less evident in vivo (239).
Macrolide antibiotics such as erythromycin, clarithromycin, and azithromycin appear to have the ability to decrease sputum production in patients with chronic respiratory infections (270,271,362–364). This has been attributed to a direct effect on the production of sputum (363) but may instead be due to inhibition of biofilm production by respiratory pathogens (266,270). Macrolides have been shown to markedly reduce the biofilm structure of microorganisms in their sessile phase (135) and to reduce the amount of virulent exotoxins (131,132). These microorganisms include S. epidermidis and P. aeruginosa. The mechanism seems to involve the suppression of a step or steps in the synthesis of monosaccharides, probably as a result of the suppression of mRNA (130,138). The use of macrolides such as erythromycin or azithromycin has been beneficial in chronic respiratory tract infections caused by P. aeruginosa (268–271).
Erythromycin.Erythromycin is a metabolic product of Streptomyces erythreus and consists of a 14-member lactone ring to which are attached two deoxy-sugars, desosamine and cladinose. The macrocyclic lactone ring is the source of the class name, macrolide. Erythromycin, like most macrolides, appears to act by binding in the ribosomal tunnel through which the nascent peptide moves and thus can be considered a peptidyltransferase inhibitor (130). Against some rapidly replicating bacteria, erythromycin exhibits in vitro bactericidal activity, but overall, it is considered to be bacteriostatic in clinical use.
Clarithromycin.Clarithromycin, like erythromycin, has a 14-member lactone ring structure that has been altered by the addition of a methoxy group at C6 of the lactone ring (133,361,362). This substitution primarily results in better oral absorption, with little effect on the spectrum of activity. In fact, erythromycin, clarithromycin, and azithromycin appear to bind to the same receptor on the bacterial 50S ribosome subunit. Clarithromycin, like the other macrolides, has species-specific bactericidal activity (239), as defined by in vitro methods of assessment where a greater than or equal to 3 log10 decrease in colony-forming units over a 24-hour period is defined as bactericidal (62). The species usually considered to be killed by macrolides are S. pneumoniae, S. pyogenes, and H. influenzae. However, the bactericidal activity of clarithromycin against susceptible strains of S. pneumoniae, as demonstrated in vitro by time-kill kinetic curves, has been found to lack correlation with results from the rabbit model for pneumococcal meningitis (239). The use of an in vitro method to measure total microbial killing has been suggested as a way to better assess the bactericidal activity of antimicrobial agents used in bacterial meningitis (228).
Azithromycin.Azithromycin is derived from erythromycin and differs in having a methyl-substituted nitrogen in its 15-membered lactone ring (362,365). This class of drugs receives its name, azolides, from the presence of the nitrogen group. Azithromycin has the same mechanism of action as erythromycin, and these two drugs bind so close to each other on the ribosome that they are considered competitive inhibitors. Azithromycin, like erythromycin and other macrolides, is a bacteriostatic agent. Azithromycin, however, has a major advantage, namely, its absorption and prolonged intracellular/interstitial fluid levels. In particular, the intracellular levels should greatly enhance the therapy of infections caused by intracellular pathogens.
Dirithromycin.Dirithromycin is a semisynthetic derivative of erythromycin that is converted during absorption and distribution to an active metabolite 9-(S)-erythromyclamine, which is the predominant agent found in plasma and extravascular tissues (366,367). This macrolide demonstrates high and prolonged tissue concentrations, allowing once-daily dosing. The mechanism of action is identical to that of the other macrolides. The result is bacteriostatic activity against logarithmically growing microorganisms and may include bactericidal activity against bacteria in a static growth phase. Dirithromycin, like azithromycin, does not inhibit cytochrome P450 enzymes and thus does not cause clinically important drug–drug interactions, although its gastrointestinal side effects are similar to those of other macrolides (362,368). Macrolides appear to be able to suppress the initiation of mRNA synthesis (180) and thereby inhibit the production of biofilm (135,138), exoenzymes (131,132), and other such virulence factors by a diverse group of pathogens, including P. aeruginosa (131,269). If this proves to be clinically effective in chronic infections, a once-daily dosing that achieves high and prolonged tissue concentrations and has no significant drug–drug interactions will be extremely useful.
Telithromycin.Telithromycin is a ketolide (371) in which the C11/C12 carbamate residue includes a butyl chain linking an imidazole ring and a pyridine ring (358–360). The most important factors in terms of the structure and activity of telithromycin are the lack of the neutral sugar cladinose in position C3 as well as a C11/C12 carbamate group, which together markedly increase the affinity of telithromycin for its microbial target, the 23S ribosomal drug-binding pocket (352,353,369). Telithromycin interacts with the 23S rRNA portion of the 50S subunit in the upper portion of the peptide exit channel close to the peptidyl transferase center and prevents the peptide chain from passing through the peptide exit channel (352,353,369). The increased affinity of telithromycin for the 23S rRNA is seen even in macrolide-resistant strains (370) and also results in concentration-dependent bactericidal activity and a prolonged post–antimicrobial effect against important respiratory tract pathogens. The microbiologic spectrum of activity for telithromycin includes S. pneumoniae, S. pyogenes, H. influenzae, Moraxella catarrhalis, Legionella species, Mycoplasma pneumoniae, and Chlamydia pneumoniae, which suggest that telithromycin will play an important clinical role in the empirical treatment of community-acquired respiratory tract infections (371). The pharmacokinetic profile of telithromycin demonstrates that this drug can be administered once daily without regard for meals and requires no dose reduction in elderly patients or those having hepatic impairment (372). Telithromycin is well absorbed after oral administration, rapidly penetrates into respiratory tissues and fluids, and is highly concentrated within white blood cells. Integration of pharmacokinetic and pharmacodynamic properties reveals that telithromycin has a high AUC:MIC ratio compared with macrolide antimicrobial agents, which results in enhanced efficacy. Resistance, although rare to date, can occur (356). Finally, telithromycin is well tolerated and has a low propensity for drug interactions.
Clindamycin.Clindamycin is a member of the lincosamides, which are chemically unrelated to the macrolides (354). Lincosamides consist of an amino acid linked to an amino sugar. Clindamycin is a 7-deoxy-7-chloro derivative of lincomycin, the first member of the lincosamide class. These two agents, like the macrolides, act on the peptidyltransferase center of the 50S subunit of bacterial ribosomes (134). Clindamycin may exhibit in vitro bactericidal activity against susceptible microorganisms such as S. aureus and B. fragilis, but its in vivo activity is considered bacteriostatic. The reason for this species-specific bactericidal activity is unknown but may reflect differences in the apoptotic mechanisms of these microorganisms.
Streptogramins.The streptogramin group of antibiotics includes the mikamycins, the pristinamycins, the oestreomycins, and the virginiamycins. These compounds are classified into two main groups: polyunsaturated cyclic peptidolides and cyclic hexadepsipeptides. Both groups possess a wide variety of chemical functions. Quinupristin/dalfopristin (Synercid) is a semisynthetic antibiotic consisting of two water-soluble streptogramin components: pristinamycin IA, a peptidic macrolactone, and pristinamycin IIA, a polyunsaturated macrolactone (373–375). These two macrolactones are modified to be water-soluble and together demonstrate synergistic and concentration-independent lethal activity against gram-positive pathogens, including S. aureus, in contrast to the individual components, which are only inhibitory (376). This bactericidal effect is seen clinically as well, as evidenced by the successful treatment of bacterial endocarditis. It is thought that this activity is related to irreversible binding to ribosomes (377). The 70S ribosomal subunit appears to be the target, with binding, closing, or narrowing the extrusion channel. This combination also demonstrates a postantibiotic effect (378). Moreover, each component diffuses throughout cardiac vegetations and is more concentrated in these vegetations than in cardiac tissue. The peptide macrolactone is distributed homogeneously throughout each vegetation, while the polyunsaturated macrolactone reaches the core, with a gradient of decreasing concentrations from the periphery.
Exposure of S. aureus to quinupristin/dalfopristin results in two major cell alterations: an increase in cell size and an increase in the thickness of the cell wall. Some cells exhibit multilayered cell walls with as many as six layers (92) (Fig. 10.4), which is considerably thicker than the two- or three-layer cell wall that has been seen with exposure of S. aureus to chloramphenicol (94). This alteration is likely to be a stringent response.
The fluoroquinolones are synthetic antimicrobial agents that inhibit bacterial topoisomerases. Within several decades, these agents have proven their usefulness as a major class of broad-spectrum agents and have therapeutic potential yet to be realized (379). The first member of this group was nalidixic acid. The newer fluoroquinolones are all structurally similar to nalidixic acid and have a common skeleton, the four-quinolone planar heterocycle nucleus (380). Most also have a fluorine atom at position C6 of the structure (hence the name fluoroquinolones). Substitution on this skeleton is greatly aided by the vast knowledge accumulated regarding the structure–activity relationships, and the optimal groups for each position, in terms of size, shape, and electrical properties, have been well defined (380–384). Specific structural features that enhance the activity of fluoroquinolones are listed in Table 10.1.
These features can be used to predict the best configuration for a specific microorganism. For example, there are a number of new antibiotics that have demonstrated activity against Mycobacterium species (385). Among these are fluoroquinolones (386,387), despite the fact that M. tuberculosis does not possess homologs of the topoisomerase IV parC and parE genes (388). These fluoroquinolones can be evaluated by predicting their active structures (389). In addition, the most beneficial substituent of quinolones today holds not only for gram-positive and gram-negative bacteria but also for Mycobacterium avium (389); an N-substitution offers the greatest enhancement (390). This may be related to two factors: the amine groups may allow a greater concentration of the uncharged species to exist at neutral pH, and the N1-cyclopropylamine may also serve as a suicide inhibitor of redox enzymes, such as cytochrome P450 and methylamine dehydrogenase, which are present in bacteria. Alkylation of the N1-substituted moiety increased the activity of these agents against mycobacteria (390).
Fluoroquinolones interact with either or both of two topoisomerases, topoisomerase II and IV (115,117,325,391,392). Topoisomerase II (DNA gyrase) tends to be the primary target in gram-negative microorganisms; topoisomerase IV tends to be the primary target for gram-positive microorganisms. DNA gyrase is a vital bacterial enzyme that catalyzes the introduction of negative superhelical twists in circular DNA. Negative superhelical twists in circular DNA are needed for the following reasons. In order for DNA replication or transcription to take place, the two strands of double-helical DNA must first be separated. Separation, however, results in excessive positive supercoiling of the DNA in front of the point of separation. The bacterial enzyme DNA gyrase prevents this positive supercoiling by introducing negative supercoils into DNA. Gyrase consists of two functional subunits, the A subunit and the B subunit. The first catalyzes the step involving the passage of a segment of DNA through a double-stranded DNA break held open by the enzyme and then reseals the break. The second is responsible for adenosine triphosphate (ATP) hydrolysis, which is required for catalytic supercoiling by gyrase. Topoisomerase IV decatenates DNA and removes positive and negative supercoils: as in the case of topoisomerase II, there are two subunits involved. Quinolones rapidly bind to the topoisomerase-DNA complexes, and the resultant quinolone-topoisomerase-DNA complex is known as a cleaved complex (235). The cleaved complex essentially traps the topoisomerase and the single-stranded DNA and results in DNA double-stand breakage, resulting in chromosome fragmentation. The formation of these cleaved complexes also results in a variety of other quinolone-mediated phenomena. Binding of quinolones to GyrA (ParC helix-4 region) of DNA gyrase, which is located near the DNA gate region, results in rapid inhibition of nucleic acid biosynthesis. In contrast, binding of quinolones to GyrB (ParE) of topoisomerase IV results in a slower inhibition of nucleic acid biosynthesis. The inhibition of nucleic acid biosynthesis correlates with the bacteriostatic drug susceptibility (i.e., the MIC) but does not correlate with rapid cell death (235). Inhibition of nucleic acid biosynthesis combined with the double-stranded DNA breaks, in turn, induces the SOS response. Induction of the SOS response leads to production of a chromosomally encoded toxin, MazF, which alters protein carbonylation resulting in oxidative stress (393). Oxidative stress may then lead to an oxidative damage cellular death pathway resulting in rapid cell death (44,45,125). This oxidative damage cellular death pathway involves generation of highly destructive hydroxyl radicals through the Fenton reaction (44,45,394). Finally, induction of the SOS response also leads to filamentation of the bacteria although the role of filamentation in cell death, if any, is yet unclear (235).
In summary, fluoroquinolones are bactericidal agents that exhibit concentration-dependent killing with little postantibiotic effect at concentrations ranging from those equal to the MIC (1 times the MIC) up to approximately 10- to 20-fold higher (10 to 20 times the MIC). At higher concentrations (greater than 10 to 20 times the MIC), the SOS response appears to be evoked (44,45,235,395). This can result in a secondary bactericidal effect that is accompanied by a longer postantibiotic effect and a decrease in the emergence of resistant clones (234,396). This secondary bactericidal effect also may involve a signal to the apoptotic mechanism of the microbial cell, ultimately leading to rapid apoptosis (44,45,235). Cell death, therefore, may be relatively slow and related to a protein synthesis–dependent process or may be rapid and related to a protein synthesis–independent process.
DNA gyrase is the target of an increasing number of antibiotic classes. One of these classes consists of the synthetic quinolones, which act by interfering with the DNA-rejoining step involving the A subunit, while another class consists of the natural aminocoumarin-type compounds, which compete with ATP for binding to the B subunit of the enzyme. A related class, consisting of the 2-pyridones, is discussed later in “Antimicrobial Classes with Potential for Future Use.”
Aminocoumarin-type antimicrobial agents are characterized by their 3-amino-4,7-dihydroxycoumarin moiety and are produced by different strains of Streptomyces (397). Aminocoumarins include structurally similar agents, novobiocin, clorobiocin, and coumermycin as well more structurally complex agents, simocyclinone and rubradirin. Novobiocin, clorobiocin, and coumermycin share common structural features, which include the 3-amino-4,7-dihydroxycoumarin moiety as well as an L-noviosyl sugar and an aromatic acyl component attached to the amino group of the aminocoumarin moiety. Novobiocin and clorobiocin differ structurally only by substitution at two positions: CH3 versus Cl at position 8′ of the aminocoumarin ring and carbamoyl versus 5-methyl-pyrrol-2-carbonyl at the 3″-OH of novoise. Novobiocin, clorobiocin, and coumermycin interact with bacterial DNA gyrase with both the aminocoumarin moiety and the substituted deoxysugar moieties binding to the B subunit of DNA gyrase (398). The carbamoyl group of novobiocin and the 5-methyl-pyrrol-2-carbonyl group of clorobiocin are important for the binding of these agents to the GryB subunit. Moreover, the binding site of the aminocoumarin-type antimicrobial agents overlaps with the binding site of ATP; therefore, these agents competitively inhibit the ATP-dependent supercoiling of DNA. Topoisomerase IV is an additional target for clorobiocin but not novobiocin. Finally, the more structurally complex aminocoumarins simocyclinone and rubradirin do not have deoxysugar moieties at the 7-OH group of the aminocoumarin structure and thus do not bind with the GryB subunit of DNA gyrase. However, simocyclinone has been shown to bind to the N-terminal domain of the GryA subunit and thus also inhibits DNA gyrase (399,400). Although rubradirin does not inhibit DNA gyrase, it is structurally similar to the ansamycin/rifamycin family of antibiotics and is a potent inhibitor of RNA polymerase (401).
Novobiocin is a glycosylated dihydroxycoumarin derivative of a natural aminocoumarin-type antibiotic produced by Streptomyces niveus. Novobiocin is the only member of the aminocoumarin-type agents that has been used clinically. Novobiocin acts on the B subunit of DNA gyrase and interferes with ATP hydrolysis (402). Novobiocin exhibits bactericidal activity in vitro and in vivo. Perhaps the greatest benefit would come from combining this agent with other DNA gyrase inhibitors (115) in order to decrease the selection of resistant mutants. However, other agents in the aminocoumarin family may be more useful in this regard.
The sulfonamides were the first effective chemotherapeutic agents to be used for the systemic therapy of bacterial infections in humans. Domagk (403) recognized this potential with Prontosil, which has sulfanilamide as its active metabolite, and later received the Nobel Prize in Medicine for this discovery. Sulfonamide is the generic name for derivatives of p-aminobenzenesulfonamide. Sulfonamides inhibit the folate pathway in prokaryotic and primitive eukaryotic cells. These agents owe their activity to their being structural analogs and competitive antagonists of p-aminobenzoic acid (PABA); this antagonism is in part due to the attachment of the sulfa molecule directly to the benzene ring. Sulfa drugs exactly fit into the PABA-binding pocket, with the negatively charged oxygen atoms of the sulfonyl group and their common phenyl groups engaging the same hydrophobic site in the PABA-binding pocket (404). The result is competitive inhibition of dihydropteroate synthase, the bacterial enzyme that catalyzes the incorporation of PABA into dihydropteroic acid, the immediate precursor of folic acid (404–406). The in vitro and in vivo effects of this inhibition are bacteriostatic.
Trimethoprim chemically is 2,4-diaminopyrimidine and was specifically synthesized as a competitive inhibitor of dihydrofolate reductase (407,408). Trimethoprim selectively inhibits bacterial dihydrofolate reductase and does not interfere with mammalian dihydrofolate reductase due to the fact that trimethoprim does not fit into the nucleotide-binding site of the mammalian enzyme (409). This antibiotic inhibits folic acid synthesis, acting on the enzyme step that immediately follows that blocked by sulfonamides. The folic acid pathway is a small portion of a more complex pathway known as the aromatic biosynthetic pathway. The aromatic biosynthetic pathway is absent in mammals; the dietary requirements of mammals for phenylalanine, tryptophan, folic acid, and vitamin K reflect this absence. Bacteria, on the other hand, rely on this pathway, which makes it an attractive target for antimicrobial agents. Trimethoprim, like the sulfonamides, is bacteriostatic when used alone. The combination of these two inhibitors of folic acid synthesis, however, acts synergistically and produces a bactericidal effect (410–413).
Chloramphenicol, also called Chloromycetin, is a broad-spectrum antibiotic produced by a variety of Streptomyces species, including Streptomyces venezuelae (414,415). This antimicrobial agent is unique among natural compounds in that it contains a nitrobenzene moiety that is connected to propanol as well as an amino group binding a derivative of dichloroacetic acid (414,416). These latter moieties, propanol and dichloroacetic acid, must be intact for antimicrobial activity to occur, although substitution of the dichloroacetamide side chain is possible. A common mechanism of resistance is the acetylation of one or both hydroxyl groups on the propanediol moiety. These hydroxyl groups, however, cannot be substituted to prevent this type of resistance. The chloramphenicol molecule is one of the simplest antibiotic structures and was quickly and easily synthesized (414,417), becoming the first antibiotic whose chemical synthesis was feasible for large-scale commercial production.
Chloramphenicol is a small, uncharged, nonpolar molecule and readily diffuses through the cell wall/membrane. Although early work suggested that chloramphenicol is actively taken up by E. coli, later studies showed that there is actually an endogenous active efflux that depends on the proton motive force (418).
Chloramphenicol inhibits the peptidyltransferase reaction in bacterial protein synthesis by reversibly binding to a site in domain V of the 23S rRNA peptidyltransferase center of the 50S subunit of the 70S ribosome (417,419). This attachment prevents the attachment of the amino acid–containing end of the aminoacyl–tRNA complex to the ribosome, thereby inhibiting the formation of a peptide bond and causing translational inaccuracy (420). The site of action is near that of the macrolide antibiotics, clindamycin, and linezolid. Chloramphenicol thus may inhibit these agents competitively. This inhibition of protein synthesis in most susceptible microorganisms results in a bacteriostatic effect, although certain microbial pathogens, such as H. influenzae, S. pneumoniae, and Neisseria meningitidis, are readily killed in vivo by clinically achievable concentrations (421). This bactericidal activity may be due to the triggering of apoptotic mechanisms in these pathogens.
Chloramphenicol has other inhibitory effects in bacterial cells that are not related to inhibition of protein synthesis. Perhaps the most important effect is that on the bacterial translocase reaction in certain microorganisms. Chloramphenicol strongly inhibits the synthesis of teichoic acid in Bacillus licheniformis by inhibiting the function of undecaprenol-P (422).
Chloramphenicol is among the few agents that retain antimicrobial activity against bacteria in the stringent response phase (233). It therefore has potential for being combined with other agents that have similar effects, such as metronidazole and the macrolides. Susceptibility testing methods, however, must reflect the nonreplicating growth phase when evaluating these combinations.
The rifamycins are a group of structurally similar macrocyclic antimicrobial agents produced by Amycolatopsis mediterranei (423,424). These agents belong to the family of ansamycin antibiotics, which are produced by various actinomycetes (423). The name ansamycin stems from the basketlike molecular architecture comprising an aromatic group bridged at nonadjacent positions by an aliphatic chain (423,424). The aromatic group for the rifamycins is a naphthalene ring system; thus, the basic structure of rifamycins is a naphthalene ring spanned by a long aliphatic loop. The target of rifamycins is bacterial RNA polymerase, which is a multi-subunit enzyme responsible for bacterial transcription (119–121,425–428). RNA polymerases are nucleotidyl transferase enzymes that are able to generate an RNA copy of a DNA or RNA template chain and thus control initiation and termination of transcription. RNA polymerases are found in nature in all eukaryotes, prokaryotes, and archaea as well as in many viruses; prokaryotic RNA polymerases differ from eukaryotic enzymes. Bacterial RNA polymerase is composed of four polypeptide subunits: α required for assembly of the enzyme, β involved in chain initiation and elongation, β′ binds to the DNA template, and Ω constrains the β′ subunit and aids its assembly into RNA polymerase. In the bacterial RNA polymerase, the large β and β′ subunits form the pincers of a crab-claw molecule; a large channel between the pincers holds the following components: the RNA 3′-OH within the active site, an 8-9-base-pair RNA-DNA hybrid at the growing end of the transcript, at least 10 base pairs of duplex DNA downstream of the hybrid and approximately six nucleotides of single-stranded RNA upstream of the hybrid (119).
Rifampin is a semisynthetic derivative with modifications at the 4-position on the naphthalene ring of one of the natural rifamycins, rifamycin B. Other semisynthetic derivatives have been developed (429). Rifampin, like other rifamycins, acts by binding to the β subunit of the RNA polymerase and sterically blocks the extension of the nascent RNA chain after the first or second condensation step (119,425–428). The final result is inhibition of protein synthesis by prevention of chain initiation. Rifampin is a bactericidal agent in vivo as well as in vitro, but because of the high rate of resistant mutants, this antibiotic is always used in combination with another agent when treating serious infections.
The tetracyclines are broad-spectrum antibiotics discovered in the late 1940s following the isolation of chlortetracycline from Streptomyces aureofaciens (430–433). These agents were the first broad-spectrum antibiotics to be widely used in the therapy of bacterial infections in humans and are still in clinical use today (430,433).
Tetracycline antibiotics consist of a hydronaphthacene nucleus with four fused rings. Substitutions on this fused ring structure at carbons 4, 5, and 6 have resulted in semisynthetic agents; two of these are doxycycline and minocycline. These semisynthetic compounds are more lipophilic than their precursors and hence are more active.
At physiologic pH, many tetracyclines can exist in a mixture of two forms, a nonionized lipophilic form and a zwitterionic hydrophilic form (431). The lipophilic form assists passage through the cell membrane, while the hydrophilic form assists diffusion through the biofilm and the cytoplasm. Passage of tetracycline itself through gram-negative outer membranes involves the porins, with a preference for OmpF. OmpF porins are cation-selective, and tetracycline may pass through these channels as a cationic chelate of magnesium. The uptake of tetracycline across the cell membrane has been shown to involve an energy-dependent process that is now thought to be the result of a pH gradient. After tetracyclines enter bacteria via diffusion through the cell wall/membrane, these agents remain as a magnesium chelate or are complexed as such within the cytoplasm. These tetracycline/magnesium-chelated complexes, once inside the cytoplasm, appear to be membrane-impermeable.
Tetracycline derivatives can be classified into two categories based on their mechanism of action. The first category is known as the traditional tetracyclines and includes tetracycline, chlortetracycline, minocycline, and doxycycline (430–432). These traditional tetracyclines target the 16S ribosomal particle of the 30S ribosomal subunit (434) and inhibit protein synthesis at this ribosomal level due to disruption of codon–anticodon interactions between tRNA and mRNA in which binding of aminoacyl–tRNA to the ribosomal acceptor site is prevented (118,431,432). This interaction appears to be reversible and is thought to account for the bacteriostatic nature of these agents.
The second category of tetracyclines is termed the atypical tetracyclines (435,436). In the atypical tetracyclines, substituents are introduced at carbon 9 without alteration of the basic structure of the classical tetracyclines, with the result that the primary target is not the ribosome but the cytoplasmic membrane (436). Examples of the atypical tetracyclines include chelocardin, anhydrotetracycline, and anhydrochlortetracycline; to date, none of the atypical tetracyclines are in clinical use (430,433,436). These modifications also allow the molecule to avoid recognition by a tetracycline efflux protein [TetA(B)]. The atypical lipophilic tetracyclines may exist primarily in the nonionized lipophilic form, which allows them to remain in the cell membranes. The interaction of these tetracycline analogs with the cell membrane is lethal, resulting in cellular lysis (435,436). This lethal effect may involve interruption of membrane-associated proton motive forces and energy metabolism (44,45,107).
Glycylcyclines are a new class of semisynthetic tetracyclines (437–441) that contain the N, N-dimethylglycylamido substituent at the 9-position of minocycline and 6-demethyl-6-deoxytetracycline (437–444). The presence of this substituent overcomes the two major mechanisms responsible for tetracycline resistance in a wide variety of bacterial pathogens: active efflux of the drug out of the cell and protection of the ribosomes by the production of cytoplasmic proteins (432,437,439,445). Glycylcyclines have a higher binding affinity for ribosomes than earlier tetracyclines, which is thought to explain why cytoplasmic ribosomal protection proteins are unable to confer resistance to glycylcyclines (445). This accordingly extends the spectrum of these new agents to include multiresistant strains, including Neisseria gonorrhoeae, and in addition significantly improves their activity (at least fourfold), compared with the activity of minocycline and tetracycline (442,446).
Nitroimidazoles and Nitrofurans
Nitroimidazoles and nitrofurans are synthetic antimicrobial agents that are grouped together because both are nitro group (-NO2)–containing ringed structures having similar antimicrobial effects (447). These antimicrobial effects require degradation of the agent within the microbial cell so that electrophilic radicals are formed. These reactive electrophilic intermediates then damage nucleophilic sites, including ribosomes, DNA, and RNA. The effect in vitro and in vivo against susceptible microorganisms is bactericidal.
Nitroimidazoles.The nitroimidazoles are organic nitroaromatic derivatives that are activated within the cytoplasm of microorganisms by electrons in order to exert their antimicrobial effect. The key to the antimicrobial activity of this class of drugs is the nitro group, which acts as a preferential electron acceptor (447,448). Electrons are needed to reduce the nitro group (-NO2) to an amine or amino group (-NH2). The reduction, however, does not proceed along the classical reduction pathway seen with nitrobenzene or its derivatives, such as chloramphenicol. Instead, the reduction leads to nitro radical anions, which undergo rapid decomposition to a nitrite ion and an imidazole radical. These short-lived electrophilic reduction products of the nitroimidazoles are produced within the microbial cell by enzymatic pathways utilized in anaerobic metabolism. Anaerobes depend on the ferredoxin-linked pyruvate oxidoreductase system as a major route of ATP generation. These pyruvate oxidoreductase pathways utilize ferredoxin, or similar electron transfer proteins, as electron acceptors from the oxidation of an enzyme-thiamine-pyrophosphate complex. The reduced ferredoxin normally transfers electrons to hydrogenase, but in the presence of nitroimidazoles, those electrons are transferred to the latter. Electrons from hydrogenase can also be transferred to nitroimidazoles. This transfer initiates the decomposition of the nitroimidazoles to radical electrophilic products. These products, most likely nitrite radicals, then react with nucleophilic protein sites and, among other effects, oxidize DNA, causing strand breaks and subsequent cell death.
Nitrofurans.Nitrofuran compounds consist of a primary nitro group (-NO2) joined to a heterocycle ring; this group of nitroheterocyclic compounds includes various 5- and 2-nitroimidazoles and 5-nitrofurans (449). Susceptible microorganisms have been shown to possess reductases that reduce nitrofurans to reactive electrophilic metabolites, and an inverse correlation exists between reductase levels and MICs (450,451). These electrophilic metabolites produce a qualitatively nonspecific attack on nucleophilic sites, including ribosomal proteins and mRNA.
Metronidazole.Metronidazole is a nitroheterocyclic compound belonging to the nitroimidazole group of antimicrobial agents, and it was the first of this group of drugs to show useful clinical activity (448). This antibiotic has two metabolic derivatives, a hydroxy metabolite with significant antimicrobial activity and an acid metabolite with relatively little activity (452). The activity of metronidazole, like other members of the nitroimidazole group, is related to the production of nitrite radicals (450–454). Metronidazole is rapidly bactericidal, and its high killing rate of anaerobic bacteria is not affected by nutrients or growth rates.
Nitrofurantoin.Members of the nitrofuran class include nitrofurantoin and furazolidone, the latter being available only in Europe. Nitrofurantoin is metabolized by bacterial reductases, resulting in electrophilic radicals that nonspecifically attack nucleophilic sites and inhibit protein synthesis. In addition, nitrofurantoin appears to have another important mechanism of action. Nitrofurantoin has been found to increase the levels of Gp4, an enzyme implicated in the induction of the stringent response (451). At the same time, nitrofurans, including nitrofurantoin, have specific interactions with ribosome sites such as S18 in the platform region of the 30S subunit, which disrupts codon–anticodon interactions and thereby prevents mRNA translation (361,451). Therefore, it is possible that nitrofurantoin acts to induce Gp4 while preventing translation of the Gp4-stimulated mRNAs for inducible enzymes (361,451). This would effectively prevent bacterial entry into the stringent response and thus impair viability. In agreement with this possible mechanism is the fact that nitrofurantoin resistance has been shown to confer a reduction in fitness in E. coli in the absence of the antimicrobial agent (455).
Glycopeptide antimicrobial agents currently include vancomycin and teicoplanin (456–458). Vancomycin was originally isolated from the fermentation broths of Amycolatopsis orientalis (459), while teicoplanin was obtained from Actinoplanes teichomyceticus (460). Both agents possess a heptapeptide backbone but differ in substituents.
Both glycopeptides achieve their antimicrobial activity by binding to the terminal amino acyl-D-alanyl-D-alanine, which prevents the transfer of cell wall components from the lipid carrier to cell wall growth points (456,461). Therefore, they share similar antimicrobial spectra and potencies, which are essentially confined to gram-positive bacteria (462). Their activities are, however, not identical. There are several explanations for this fact. The two agents differentially bind to other peptide components of preformed peptidoglycan, which has no direct effect but reduces the available drug. In addition, structure–activity relationships for these two glycopeptide agents (vancomycin and teicoplanin) suggest dimerization of these antibiotics as a potentially important factor in their activity. Such dimerization has been shown to enhance the binding affinities of most glycopeptides for the peptidyl-D-alanyl-D-alanine sequence present in the growing cell wall, the notable exception being teicoplanin (463). Teicoplanin is unique among the glycopeptides in that it demonstrates no measurable propensity for dimerization (464). A dimerized agent has enhanced activity because the second binding event is essentially intramolecular, whereas the activity of an agent with a lipid anchor requires two steps for the same effect. Although perhaps subtle, this difference may account for some of the difference in the activities of these two agents against certain species of enterococci. The fatty acid chain that teicoplanin carries may diminish this difference by acting as a membrane anchor, thus increasing the affinity of the antimicrobial agent for the growing cell wall.
Vancomycin.Vancomycin is a narrow-spectrum antimicrobial agent that is mainly active against gram-positive cocci (465). It is a large, complex, tricyclic antibiotic with a molecular mass of 1,449 Da. Within the chlorine face of this large molecule, there is a pocket into which the D-alanyl-D-alanine precursor of the cell wall peptidoglycan is complexed, preventing polymerization of undecaprenyl pyrophosphoryl-N-acetylmuramyl-pentapeptide (UDP-MurNAc-pentapeptide) and N-acetylglucosamine into peptidoglycan (466). The in vitro and in vivo results of this inhibition of peptidoglycan synthesis, like that caused by a β-lactam agent, are bactericidal. However, it is important to appreciate that this bactericidal effect of vancomycin (and teicoplanin) is much slower than that of the β-lactam agents (467,468). Vancomycin, therefore, should be substituted for antistaphylococcal penicillins such as nafcillin only when absolutely necessary, as in cases of penicillin allergy or methicillin resistance (465).
Teicoplanin.Teicoplanin differs chemically from vancomycin in a number of ways (469). First, teicoplanin has different carbohydrate substituents: D-glycosamine and D-mannose versus D-glucose and vancosamine in vancomycin. Next, teicoplanin has two dihydroxyphenylglycines rather than aspartic acid and N-methylleucine. Finally, teicoplanin is unique among the glycopeptides in having an acyl substituent, which is a fatty acid. This fatty acid makes teicoplanin much more lipophilic than vancomycin, accounting for its greater tissue and cellular penetration. This same property accounts for its activity against M. tuberculosis. The actual mechanism of action is identical to that of vancomycin, although the activity of these two agents is not always identical. Like vancomycin, teicoplanin is slowly bactericidal, in comparison with antistaphylococcal penicillins.
Lipoglycopeptides.Lipoglycopeptides are semisynthetic derivatives to the glycopeptides (vancomycin and teicoplanin) that contain the heptapeptide core that is common to all glycopeptides (470,471). This heptapeptide core allows members of the lipoglycopeptide family to inhibit transglycosylation by binding to D-alanyl-D-alanine stem termini in gram-positive bacteria in a manner identical to the glycopeptides. Lipoglycopeptides also possess hydrophobic and lipophilic substituents that help anchor the drug as well as target the bacterial cell membrane (107), resulting in membrane depolarization. It is the lipophilic side chains that distinguish these agents from glycopeptides such as vancomycin and also has resulted in these agents being categorized as lipoglycopeptides. The lipophilic side chains also prolong the half-life of these agents and help to anchor the drugs to the cell membrane. This dual mechanism of action results in more rapid bactericidal activity as well as activity against dormant bacteria (472). Members of the lipoglycopeptides include telavancin (approved for clinical use), oritavancin, and dalbavancin.
Telavancin and Oritavancin.Telavancin and oritavancin are members of the lipoglycopeptide class of antimicrobial agents; telavancin is in clinical use, whereas oritavancin is under development. Telavancin contains the heptapeptide core of vancomycin but possesses a hydrophobic (decylaminoethyl) side chain appended to the vancosamine sugar and a hydrophilic (phosphonomethyl aminomethyl) group on the 4 position of amino acid 7 (473). Oritavancin also contains the heptapeptide core of vancomycin but possesses a hydrophobic (N-r-[4-chlorophenyl]benzyl) group on the disaccharide sugar, the addition of a 4-epi-vancosamine monosaccharide to the amino acid residue in ring 6, and the replacement of the vancosamine moiety by 4-epi-vancosamine (474). These substitutions allow both agents to target both cell wall synthesis and cell membrane function. There are, however, some differences in the antimicrobial activity of these two lipoglycopeptides (472). Telavancin exhibits concentration-dependent bactericidal activity with the AUC/MIC being the pharmacodynamic parameter that best describes its activity. Oritavancin appears to exhibit concentration-dependent bactericidal activity in vitro and both concentration- and time-dependent bactericidal activity in vivo. Like telavancin, the AUC/MIC is the pharmacodynamic parameter that best describes the activity of oritavancin. Secondary binding of oritavancin to the pentaglycyl (Asp/Asn) bridging segment also occurs and contributes to oritavancin’s activity against vancomycin-resistant organisms. Telavancin is active against vancomycin-intermediate Staphylococcus aureus (VISA) but has poor activity against vancomycin-resistant Staphylococcus aureus (VRSA). Oritavancin is active against both VISA and VRSA. Both telavancin and oritavancin are active against VanB vancomycin-resistant enterococci; enterococci exhibiting the VanA phenotype are resistant to telavancin while oritavancin retains activity. Of note is that these structural differences also result in differences in the pharmacokinetics of these two agents. Telavancin has a half-life of approximately 8 hours and requires daily dosing, whereas oritavancin has a half-life of almost 400 hours, which may allow for one dose per treatment course.
Fosfomycin is a phosphoenolpyruvate analog that irreversibly inhibits phosphoenolpyruvate transferase. This agent is a unique antimicrobial agent unrelated to any other recognized class and is the only member, to date, of the phosphonomycin class (475). Fosfomycin is a broad-spectrum antibiotic produced by some strains of Streptomyces and by Pseudomonas syringae. The structure of fosfomycin is characterized by an epoxide ring and a carbon–phosphorus bond. This agent has an extremely low molecular mass of 138 Da and has been found to be nonreactive with negatively charged glycocalyx such as that produced by P. aeruginosa. Fosfomycin enters bacterial cells by active transport through the L-glycerophosphate and the hexose-6-phosphate uptake systems. Within the cell, fosfomycin blocks peptidoglycan synthesis through inhibition of the bacterial enzyme N-acetylglucosamine-3-O-enolpyruvyl transferase, which prevents the formation of N-acetylmuramic acid, an essential element of the peptidoglycan cell wall (203). The in vitro and in vivo results of this inhibition of polypeptide chain elongation are bactericidal in a time-dependent manner (475).
Fusidanes are a family of naturally occurring tetracyclic triterpenoid antibiotics, of which fusidic acid is the only therapeutic representative. Fusidic acid is derived from the fungus Fusidium coccineum. This antibiotic exhibits a steroidlike structure but has no steroidlike activity due to the stereochemistry of the molecule. The mechanism of action for this antimicrobial agent appears to involve binding to elongation factor G, thus inhibiting polypeptide chain elongation (476). The in vitro and in vivo results of this inhibition of polypeptide chain elongation are slowly bactericidal (477). Fusidic acid is most active against S. aureus, including methicillin-resistant isolates (477,478). The MICs for methicillin-resistant isolates range from 0.03 to 1.0 g/mL. Fusidic acid was introduced into clinical practice in 1962, and despite more than three decades of limited use, the resistance rate is still low (479). However, S. aureus is able to develop resistance to fusidic acid when this agent is used alone; resistance is thought to be due to point mutations in the fusA gene (480). Fusidic acid is not available in the United States for general use, although efforts are being made to make this agent available (481,482).
Polymyxins and Colistin
The polymyxins are a group of polypeptide antibiotics that have molecular masses of approximately 1,000 Da and are isolated from different strains of Bacillus (483). These antimicrobial agents are characterized by a heptapeptide ring, a high content of diaminobutyric acid, and a side chain ending in fatty acid residues. Only polymyxin B and colistin (polymyxin E) are currently used clinically; colistin has found increasing use due to multidrug-resistant gram-negative bacterial infections (261,484). All of the polymyxins are cationic detergents that disrupt biofilm and interact with the phospholipids of the bacterial cell membrane resulting in altered bacterial cell membrane permeability, leakage of intracellular contents, and bacterial cell death. Colistin (polymyxin E) is a polymyxin B nonapeptide derivative that lacks the fatty acid tail. Polymyxin B covalently attached to agarose inhibits the respiration and growth of gram-negative bacteria but not gram-positive bacteria. Also, spheroplasts of E. coli are seen after exposure to immobilized polymyxin B (167,344,345,483). This is interpreted as indicating the activity is directed at the outer cell membrane. The effects of immobilized polymyxin B are identical to the effects of EDTA (129). The target, like that of EDTA, may involve biofilm via displacement of magnesium and calcium ions. This effect can be reversed by an excess of these divalent cations (166,175).
Cyclic lipopeptides are a new class of potent antimicrobial agents with remarkable structural diversity (485). These agents are natural products produced by a variety of soil bacteria. Cyclic lipopeptides have nonribosomally synthesized peptide cores consisting of 11 to 13 amino acids that form a rigid 10-membered ring with an N-terminal fatty acid; this terminal fatty acid facilitates insertion into the lipid bilayer of bacterial membranes. Targeting the bacterial membrane of dormant bacteria is a relatively new approach to treating persistent infections (107). Structural diversity is a result of these agents containing multiple nonproteinogenic amino acids as well as different lipid tails. Among the members of this new class of antimicrobial agents are daptomycin, amphomycin, asparocin, friulimicin, glycinocin, laspartomycin, parvuline, and tsushimycin. Of these, only daptomycin is in clinical use (486).
Daptomycin is a semisynthetic cyclic lipopeptide antibiotic that is the first of this class to enter clinical use (486,487). The A21978C lipopeptide complex to which daptomycin belongs is produced through the action of nonribosomal peptide synthetases in Streptomyces roseosporus (488). Daptomycin appears to dissipate membrane potential (487,489). The evidence for this is the activity of daptomycin on L forms of Staphylococcus aureus, which results in the leakage of intracellular potassium. In addition, scanning electron microscopy has shown gross morphologic alterations in the cytoplasmic membrane (490) that are consistent with disruption of the cell wall/membrane (166,167,174,345). Several reports have noted that daptomycin is capable of calcium-dependent interactions with bilayer membranes (191,192). Specifically, daptomycin in the presence of calcium ions oligomerizes to form a micelle-like amphipathic structure with the hydrophobic decanoyl side chain facing inward, which creates a pseudo–positively charged surface with increased affinity for the negatively charged microbial cell membrane (493). Daptomycin also has been shown to cause reorganization of the microbial cell membrane architecture and thus cause mislocalization of essential cell division proteins (494). These observations (493,494) suggest that daptomycin directly inserts into the bacterial cell membrane. Daptomycin demonstrates bactericidal action against both stationary-phase and nondividing Staphylococcus aureus cells, suggesting that it has a direct effect on the bacterial cell membrane (236). Daptomycin thus may exert its rapid bactericidal effect (468) by dissipating the transmembrane electrochemical potential of the cell membrane. This rapid killing may be due to activation of apoptotic mechanisms that are triggered following disruption of the bacterial cell membrane potential (44,45). A similar phenomenon of dissipation of membrane potential has been described for gentamicin and Staphylococcus aureus (209).
Oxazolidinones are a new class of synthetic bacterial protein synthesis inhibitors (495,496). These agents are multicyclic compounds, some with fused rings, which represent a new series of antimicrobial agents unrelated by chemical structure to any other currently available antibiotics. Like the fluoroquinolones, another class of multicyclic compounds with fused rings, oxazolidinones are synthetic agents that offer many substitution sites for chemical modifications, as dictated by structure–activity relationships. Also, like the fluoroquinolones, some oxazolidinones have D- and L-isomers, only the latter of which is active against microorganisms (497). Salient structural features of representative agents in this class include tricyclic fused rings, which exhibit potent activity against MRSA and S. epidermidis; an appended thiomorpholine moiety, which confers potent in vitro activity against M. tuberculosis; fluorine substitution on the generic aromatic ring, which enhances activity against gram-positive cocci; and the addition of a piperazine ring, which also increases potency against gram-positive cocci (497,498). The spectrum of activity of oxazolidinones includes a diverse group of microorganisms such as Enterococcus spp, M. tuberculosis, and Bacteroides spp (495,498,499).
The mechanism of action is inhibition of protein synthesis because oxazolidinones inhibit ribosomal protein synthesis in a cell-free system (498,500). Like many antimicrobial agents that inhibit protein synthesis, oxazolidinones are bacteriostatic. However, their inhibition of protein synthesis is somewhat novel in that the oxazolidinones do not inhibit the peptide elongation step (498). Instead, the initial step of protein synthesis is inhibited, which in turn leads to codon–anticodon interactions where translation of mRNA for inducible enzymes is inhibited. In this respect, oxazolidinones are similar in action to lincosamides (134,354). This effect over time may result in cell death; both oxazolidinones and lincosamides are slowly bactericidal to certain microorganisms.
Resistance to oxazolidinones can occur by a single-step selection process, but this occurs at a frequency of less than 1 in 109. Resistance, when seen, is not associated with cross-resistance to other classes of antimicrobial agents.
Finally, oxazolidinones can be administered by both intravenous and oral routes. Peak levels in humans given 1 g orally reach 6 to 7 µg/mL, while the half-lives range from 2.4 to 12 hours (496). Oxazolidinones are metabolized by free radicals as well as excreted in the urine, with 40% to 60% of the drug intact.
Linezolid.Linezolid is the first member of the oxazolidinones in clinical use and is indicated in the therapy of nosocomial pneumonia and uncomplicated and complicated skin infections caused by select gram-positive bacteria (495,501,502). Linezolid is also active against M. tuberculosis (503), including multidrug-resistant strains (504) and has been used successfully for treatment of chronic, extensively drug-resistant TB (505). Linezolid is a morpholinyl analogue of the piperazinyl oxazolidinone and has a fluorine substitution at the phenyl 3-position (501). Although early studies suggested that linezolid inhibited protein synthesis (498), a more detailed explanation of this mechanism of action has only recently been described (506,507). The results of these studies suggest that linezolid binds in the A-site pocket at the peptidyltransferase center of the 50S ribosomal subunit. This A-site pocket is located within domain V of the 23S rRNA peptidyltransferase center near the interface with the 30S ribosomal subunit. Binding of linezolid to this A-site pocket thus interferes with the correct positioning of the aminoacyl–tRNA on the ribosome and blocks the initial step of protein synthesis. Lincosamides also inhibit the initiation of peptide chain formation (134,354), whereas the effect of the other macrolides is to prevent the extension of the growing peptide chain. It may be that blocking the initial step of protein synthesis accounts for the slowly bactericidal activity of both lincosamides such as clindamycin and linezolid.
Diarylquinolines are a new class of antimicrobial agents that differ structurally and mechanistically from fluoroquinolones and other quinolone classes (508).
Antimicrobial Classes with Potential for Future Use
Diarylquinolines are a new class of antimicrobial agents that differ structurally and mechanistically from fluoroquinolones and other quinolone classes (508). Although this class has a nitrogen-containing heterocycle nucleus that resembles the quinolone nucleus, it differs mainly in the specificity of the novel functionalized lateral 3′ chains. Whereas fluoroquinolones target topoisomerases, diarylquinolines target microbial ATP synthase (508–511). Targeting respiratory ATP synthase, a component of the microbial energy metabolic pathway (107), is unique and interferes with the dormant phase of microbial pathogens as well as with the replicating phase (511). Diarylquinolines initially were developed for therapy against M. tuberculosis due to their activity against dormant strains (193,512). The first member of the diarylquinoline class, bedaquiline, has been successfully used for TB in clinical trials (25,513) and has been approved for use in the United States. Although bedaquiline does not have activity against bacteria other than mycobacteria, chemical derivatives with a core structure similar to bedaquiline have been developed and tested against gram-positive pathogens such as Streptococcus pneumoniae and Staphylococcus aureus with promising results (508).
The 2-pyridones are a new class of broad-spectrum antimicrobial agents that inhibit bacterial DNA gyrase (116,514,515). These compounds are similar to fluoroquinolones but differ by placement of the nitrogen atom in the ring juncture. Because the basic ring structure of the molecule is different from that of the quinolones or naphthyridines, 2-pyridones appear to bind differently at the DNA gyrase site. Accordingly, these agents have been found to be active against fluoroquinolone-resistant bacteria (116,516). The 2-pyridones exhibit in vitro bactericidal activity. Like fluoroquinolones, these agents are water-soluble and thus have excellent bioavailability when taken orally. Hundreds of 2-pyridones have been synthesized and evaluated in vitro and in vivo, with selected agents now moving toward human clinical trials (116,515). Moreover, triazoles have been introduced in position 8 and 2 of ring-fused bicyclic 2-pyridones (517). Several of these triazole functionalized ring-fused 2-pyridones are being evaluated for in vitro antibacterial properties.
Lantibiotics are a diverse group of ribosomally synthesized antimicrobial peptides produced by gram-positive bacteria (518). These small (19 to 38 amino acids) peptides are complex polycyclic molecules that contain the thioether amino acids lanthionine and/or 3-methyllanthionine (518–521). Moreover, such ribosomally synthesized polycyclic molecules are produced by macrocyclization, which is a common method used in nature to constrain the conformational flexibility of natural peptides of both ribosomal and nonribosomal origin (522,523). There are at least three genetic pathways to lanthionine-containing peptides, and these are widespread in nature (523). Lantibiotics are produced by a wide range of gram-positive bacteria, including a variety of lactic acid bacteria (518,524,525). Lantibiotics are primarily active against gram-positive bacteria (519,526) via a number of diverse mechanisms (518,527,528). Some of these peptides—class 1 lantibiotics such as nisin, subtilin, and Pep5—are positively charged, amphiphilic molecules that exert their primary bactericidal action on the cell membrane by causing pores (518,519,521,527). Nisin has a unique pore-forming activity in that it uses the cell wall precursor lipid II as a docking molecule (524). Nisin also appears to have a dual mechanism of action due to binding to the cell wall precursor lipid II as this binding also inhibits cell wall biosynthesis (528). Class 2 lantibiotics such as cinnamycin and the related duramycins possess globular shapes and no net charge or a negative charge. These globular lantibiotics appear to inhibit phospholipases by binding phosphoethanolamine (518,520,521,527). Class 2 lantibiotics also include mersacidin; this lantibiotic does not form pores in plasma membranes but instead inhibits peptidoglycan synthesis, probably on the level of transglycosylation by complexing lipid II (518,520,521,527,529,530). Some class 2 lantibiotics appear to be bacteriostatic, whereas others appear to be bacteriocidal; this may be related to whether the lantibiotic possesses a dual mechanism of action or not. A third class of lantibiotics has been proposed (518); these lantionine-containing peptides lack significant antibiotic activity but instead have other functions for the producing cell. These lantibiotics include SapB, AmfS, and SapT (518).
Cationic peptides, also called defensins, are short (20 to 50 amino acids) amphiphilic polycationic peptides that function as an important mechanism of innate immunity in plants and animals (531,532). These antimicrobial peptides are produced by phagocytic cells and lymphocytes as well as by the epithelial cell lining of the gastrointestinal and genitourinary tracts, the tracheobronchial tree, and keratinocytes (533). There are more than 800 sequences of antimicrobial peptides from the plant and animal kingdoms (534). Examples of these agents include cecropins (535), melittin (536), magainins (537), and epidermin (538). Defensins such as human α-defensin are known to have antimicrobial activity (532,539). In addition to direct antimicrobial activity, these “host-defense peptides” have important immune modulatory functions that include recruitment and activation of immune cells, neutralization of lipopolysacharides, and enhancement of bacterial clearance (533,540,541). Some antimicrobial peptides are multifunctional and can enhance both cellular (Th1-dependent) and humoral (Th2-dependent) cytokine production and immune responses (533). The main targets of these antimicrobial cationic peptides are the bacterial cell membranes, with cell death resulting from interference of membrane-bound processes as well as from increased permeability. The antimicrobial activity of cationic peptides is due to physiochemical properties related to their amphiphilic nature, which allows these peptides to adopt conformations in which polar and positively charged amino acids orient to one side, whereas apolar structures orient to the other side. This arrangement enables these peptides to bind to negatively charged membrane surfaces and then integrate into the cytoplasmic membrane (532). Damage to the cytoplasmic membrane and/or pore formation may result in increased permeability (539,542). In addition, membrane-bound processes such as electron transport may be disrupted. Another effect of these cationic peptides, like that of aminoglycosides, may be the disruption of the biofilm caused by these agents displacing Mg2+ and Ca2+. Evidence for this is found in the fact that a number of these antimicrobial peptides have been noted to be active as insoluble complexes (343). This suggests that surface activity on the targeted microorganism is sufficient for lethal activity. Moreover, the interaction of antimicrobial peptides with biofilm is well served by the amphiphilic nature of these agents, which allows both amphipathic and hydrophobic portions and facilitates the passage of the molecule from the aqueous phase to the biofilm phase, where it displaces magnesium and calcium cations.
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