Bacteria versus Antibacterial Agents: an Integrated Approach
Author: Oreste A. Mascaretti1Category: Bacterial Pathogenesis
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Designed as an introductory text on antibacterial agents, this new volume provides a clear, comprehensive and concise overview of the subject, including the most recent and significant developments in antibacterial agents with novel modes of action.
The basics of bacterial structure and function are reviewed, describing the basis for understanding mechanisms of antibacterial action, as well as the mechanisms developed by bacteria to overcome the action of antibacterial agents. The volume also covers the characteristic features of bacterial pathogenicity, the genetic basis of resistance to antibacterial drugs, the biochemical mechanisms of action of antibacterial drugs, how antibacterial drugs reach their targets in gram-positive and gram-negative bacteria, and the wide range of human immune responses against bacterial infections.
Recent advances in research and development of new classes of antibacterial drugs are examined, as are improved analogs of penicillin, cephalosporins, carbapenems, aminoglyosides, tetracyclines, etc.
Hardcover, 393 pages, illustrations, index.
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Chapter 1 : Structure and Function of Prokaryotic and Eukaryotic Cells
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This chapter provides an overview of the basic concepts of eukaryotic and prokaryotic cell structure and function. The practical importance of each topic is described in the context of understanding the invasion of eukaryotic hosts by pathogenic bacterial cells. Eukaryotic cells are generally 10 to 100 µm in diameter and thus have 103 to 106 times the volume of typical prokaryotic cells. Organelles commonly found in animal cells include the nucleus, the endoplasmic reticulum, the Golgi apparatus, lysosomes, the mitochondria, peroxisomes, and ribosomes. The outer membrane of gram-negative bacteria provides structures and receptors that affect adhesion to host cells, resistance to phagocytosis, and susceptibility to bacteriophages. Bacteria occur as single cells or as cell associations. The bacterial cell wall is a unique structure which surrounds the cytoplasmic membrane. Bacterial cell walls are constructed from a variety of macromolecules and polymers. Structurally, the wall is necessary for maintaining the cell’s characteristic shape and countering the effect of osmotic pressure. Peptidoglycan (or murein) is a cross-linked biopolymer. The nucleoid is the site of DNA and RNA synthesis. The chromosome is the main genomic element of bacteria as a single larger circular DNA molecule. It contains the genes for all "essential" functions and structures of the bacterial cell.
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Chapter 2 : Bacterial Pathogenesis
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This chapter serves as an introduction of some of the most relevant points and highlights features that are particularly essential for an understanding of bacterial invasion of eukaryotic cells, immunity to extracellular and intracellular bacteria, evasion of immune mechanisms by extracellular and intracellular bacteria, antibacterial mechanisms of action, and bacterial resistance to antibacterial agents. The chapter provides definitions of some important terms in bacterial pathogenesis such as bacterial pathogen, virulence factors, chaperone, lectins and enterotoxins. Adhesins are assembled into pili or fimbriae that extend from the bacterial surface. Alternatively, the adhesins are directly associated with the microbial cell surface. These adhesions can be performed by the afimbrial adhesins (also called nonfimbrial adhesins). The observation that culture supernatant free from bacteria fully reproduced the symptoms of diseases such as diphtheria, tetanus, cholera, and botulism led to the conclusion that in these instances, bacterial toxins were the only factors needed by bacteria to cause a disease. In the chapter, various aspects of bacterial pathogenicity have been presented from the point of view that these microorganisms exist as isolated single cells suspended in an aqueous environment (i.e., the planktonic mode); however, most in vivo populations of bacteria grow as adherent bacterial biofilms. Inhibition of virulence factors is one potential therapeutic strategy in the search for novel targets for new antivirulence drugs such as vaccines and inhibitors of bacterial adhesion and of LPS synthesis.
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Chapter 3 : The Immune System: an Overview Immunodeficiencies Immunocompromised Hosts Innate Immunity
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This chapter presents the basic principles of the immune response of the host through the innate and adaptive immune systems, particularly as they relate to bacterial infection. Host-bacterium relationships may be altered by antibacterial drugs in three major ways: alteration of the tissue response, alteration of the immune response, and alteration of the microbial flora. The immune system consists of innate immunity and adaptive immunity. The function of the complement system (C3b) is discussed in the chapter. The major antigen-presenting cells (APCs) populations in the immune system are composed of mononuclear phagocytes and dendritic cells. Antibodies (immunoglobulins) are a group of glycoproteins present in the blood serum and tissue fluids of mammals, which can combine with antigenic determinants. Secretory immunoglobulin A (IgA) is the primary antibody of the secretory immune system. This system is found in the gastrointestinal tract, upper and lower respiratory tracts, and genitourinary system. Secretory IgA is also found in saliva, tears, and breast milk. Phagocytes have an intrinsic ability to bind directly to microorganisms via nonspecific cell surface receptors, form phagosomes, and digest the microorganisms. This phagocytic process can be greatly enhanced by opsonization by the complement component C3b or by both antibody and C3b. In order to be cured of a bacterial infection, a patient must have an operative immunological defense mechanism (i.e., must be immunocompetent). If antibacterial therapy is given, it will help fight invading bacteria; however, antibacterials alone, without the contribution of the immune response, are seldom able to overcome an infection.
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Chapter 4 : Molecular Genetics of Bacteria
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This chapter provides an outline of molecular bacterial genetics as an aid to understanding the origins and nature of bacterial resistance to antibacterial agents through mutation, bacterial recombination, and transfer of bacterial plasmids and transposable elements. Also, the three types of bacterial gene transfer (transformation, transduction, and conjugation) are discussed. Gene expression is accomplished through a sequence of events in which the information contained in the base sequence of DNA is first transcribed into an RNA molecule, which is used to determine the amino acid sequence of a protein molecule. An example of an alkylating mutagen is 1-methyl-3-nitro-1-nitrosoguanidine, which adds a methyl group to the oxygen atom at position 6 of the guanine molecule causing it to mispair with thymine. The chapter provides a general overview of bacterial recombination and on the introduction of both bacterial plasmids and transposable elements. Transduction may be the most common mechanism for gene exchange and recombination in bacteria. The spread of bacteria with resistance to diverse antibacterial agents is one of the most serious threats to the successful treatment of bacterial infections.
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Chapter 5 : Mechanisms of Bacterial Resistance to the Action of Antibacterial Agents
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Over the past 63 years (1940 to 2003), the emergence and spread of bacterial resistance to the action of antibiotics and synthetic antibacterial agents are certainly the most striking examples of evolution that have arisen in bacteria. Bacterial resistance is described in terms of either phenotypic (e.g., growth patterns) or genotypic (e.g., presence or expression of genes) characteristics of bacteria, or both, and can be categorized according to origin (intrinsic versus acquired resistance) or type (single or multiple). Conjugative transposons of gram-positive as well as gram-negative bacteria represent another efficient mode of transfer of antibacterial resistance genes between phylogenetically distant bacteria genera. Horizontal gene transfer among bacteria is a perpetual phenomenon that has a significant impact on bacterial evolution. This chapter presents a general overview of the major mechanisms of bacterial resistance. Impermeability was considered to be the main mechanism of tetracycline resistance in gram-negative bacteria, due to less drug accumulation in resistant cells. The β-lactamase genes are located in the chromosome, in plasmids, or in transposons. The major mechanism of inactivation of aminoglycoside antibiotics involves aminoglycoside-modifying enzymes. Chloramphenicol acetyltransferase inactivates chloramphenicol by acetylating it using acetylcoenzyme A as the acetyl group donor. A family of enzymes is known to catalyze the mono-or dimethylation of the N-6 amino group of adenine in a highly conserved region of 23S rRNA, which may be involved directly in the formation of peptidyltransferase centers.
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Chapter 6 : Antibiotics and Synthetic Antibacterial Agents
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In this chapter, the antibiotics and synthetic antibacterials are grouped on the basis of their chemical structure and by mechanism of action. Substructure groups are the penicillins, the cephalosporins, the carbapenems, the monobactams, and the β-lactam derivatives clavulanic acid, sulbactam and tazobactam, which are β-lactamase inhibitors. Fosfomycin [L-(cis)-1,2-epoxypropylphosphonic acid] (formerly known as phosphonomycin) is a naturally occurring antibiotic obtained from species of Streptomyces. The nitrofurans are a class of synthetic antibacterial agents characterized by the presence of a 5-nitro-2-furanoyl group. Some of the antibiotics that interfere with the biosynthesis of peptidoglycan are β-lactams (penicillins, cephalosporins, cephamycins, monobactams, and carbapenems), glycopeptides (vancomycin and teicoplanin), D-cycloserine, fosfomycin, and bacitracin. The bactericidal activity of the polymyxins and cationic antimicrobial peptides results from their interaction with the bacterial cytoplasmic membrane, causing gross disorganization of its structure. For growth and multiplication, microorganisms conduct a variety of biochemical and metabolic processes to obtain energy and new cell material. All industrial microbiology processes require the initial isolation of microorganisms from nature. Antibiotic-producing microorganisms are subjected to extensive genetic manipulations and modifications before they are used for antibiotic-manufacturing purposes. Nonpolar antibiotics are usually purified by solvent extraction procedures; water-soluble compounds are commonly purified by ion-exchange methods or chemical precipitation.
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Chapter 7 : β-Lactams, Penicillin-Binding Proteins, and β-Lactamases
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Several properties shared by some of the β-lactam antibiotics are spectral characteristics of the β-lactam group; however, in other cases the properties are very different, and it is difficult to give a clear picture of properties of the individual members and how they differ from each other. Penicillin-binding protein (PBPs) are a group of bacterial membrane-bound enzymes whose active sites are available in the periplasmic space. The production of β-lactamases is considered to be the most common mechanism of bacterial resistance to β-lactam antibiotics. According to Ambler, β-lactamases are also grouped into four molecular classes based on their primary sequence homology. Serine β-lactamases differ from serine DD-transpeptidases in that they catalyze the deacylation step very efficiently only with β-lactams that have an aromatic (planar) substituent joined to the secondary amide side chain. The amino acid alignments reveal several conserved boxes that consist of strict identities or homologous amino acids. The significance of the homologies and differences is highlighted by the recent results of X-ray crystallography and site-directed mutagenesis experiments that have demonstrated the three-dimensional structural similarities between representatives of β-lactamases enzymes. Structural studies suggested that the conserved residue Tyr150 is the catalytic base that activates the hydrolytic water for its attack on the acyl intermediate.
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Chapter 8 : Inhibitors of Peptidoglycan Biosynthesis
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This chapter provides an overview on inhibitors of peptidoglycan biosynthesis. Derivatives of penicillanic acid comprise two groups of therapeutic agents of considerable importance in medicine: the antibacterial penicillins and the β-lactamase inhibitors. Penicillin G (benzylpenicillin) and penicillin V (phenoxymethylpenicillin) are the only penicillins used in their natural forms as obtained from the fermentation of Penicillium chrysogenum. The other penicillins in clinical use are derived from 6-β-aminopenicillanic acid using standard synthetic procedures. The structural modification in the side chain of antistaphylococcal β-lactamase-resistant semisynthetic penicillins provides enough steric hindrance to make them very poor substrates for most β-lactamases. Nafcillin and the isoxazolyl penicillins also are used only for treatment of infection due to β-lactamase-producing staphylococci. The aminopenicillins include ampicillin, its ethoxycarbonyloxyethyl ester (bacampicillin), and amoxicillin. The antipseudomonal penicillins are semisynthetic derivatives of penicillanic acid and are categorized into two subgroups: the carboxypenicillins, which include carbenicillin indanyl ester (an ester for oral administration) and ticarcillin, and the ureidopenicillins, which include mezlocillin and piperacillin. Antipseudomonal penicillins retain most of the antibacterial activity of the aminopenicillins but have added activity against gram-negative bacilli. Indanyl carbenicillin (with a carboxyl side chain) is well absorbed from the gastrointestinal tract and is hydrolyzed by esterases to carbenicllin, the active drug. In the treatment of moderate to severe Pseudomonas infection, an aminoglycoside of the antipseudomonal group (gentamicin, tobramycin, or amikacin) is often administered along with an antipseudomonal penicillin (mezlocillin or piperacillin).
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Chapter 9 : Inhibitors of Peptidoglycan Biosynthesis
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This chapter provides an overview on inhibitors of peptidoglycan biosynthesis cephalosporins. The clinical use of cephalosporin C was limited by its generally weak antibacterial activity. With the penicillins as a precedent, the 7-acylamino group was the first target for variation in the search for improved activity. The first-generation cephalosporins are used to treat Staphylococcus aureus and nonenterococcal streptococcal infections when it is necessary to avoid the use of penicillin. The second-generation cephalosporins should be considered in three groups: the true cephalosporins, the cephamycins, and the carbacephems. The first is a syn-oxime, found in cefuroxime as well as in other third- and fourth-generation cephalosporins. The effectiveness against gram-negative bacteria that is so notable in carbenicillin and ticarcillin (which is attributed to the α-COOH group on the acyl side chain) is also present in moxalactam. As in amoxicillin, the p-OH group on the benzene ring is incorporated in moxalactam to increase the level of drug in blood and to increase its half-life. The 1-methyl-tetrazolyl group at position 3, which has been useful in several third-generation cephalosporins, was also incorporated into the structure of this molecule. The first-generation cephalosporins have a spectrum that includes E. coli, K. pneumoniae, P. mirabilis, and most gram-positive cocci, although not enterococci or methicillin-resistant enterococci. The second-generation cephalosporins have broader in vitro activity against gram-negative bacteria. The third-generation cephalosporins are more active than first- and second-generation drugs against gram-negative organisms.
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Chapter 10 : Inhibitors of Peptidoglycan Biosynthesis
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The carbapenems are a subgroup of β-lactam antibiotics with a common carbapenem nucleus. Carbapenems are broad-spectrum antibiotics with activity against gram-positive and gram-negative bacteria. Like other β-lactam antibiotics, imipenem and thienamycin interfere with the biosynthesis of the peptidoglycan polymer. The discovery of the excellent activity of the imipenem-cilastatin combination against a wide variety of aerobic and anaerobic microorganisms led to extensive work on the isolation of other naturally occurring carbapenems by pharmaceutical laboratories worldwide as well as on the production of synthetic compounds. In vitro, meropenem, like imipenem, is active against most clinically important gram-positive and gram-negative aerobes and anaerobes. Both imipenem and meropenem are inactive against methicillin-resistant staphylococci, Enterococcus faecium, or Stenotrophomonas maltophilia. Ertapenem is a new β-lactam antibiotic belonging to the carbapenem subgroup. It possesses a 1-β-methyl group on the carbapenem nucleus. Unlike the penicillins, cephalosporins, and carbapenems, which are bicyclic compounds, the monobactams are monocyclic β-lactam antibiotics. The first monobactams to be discovered were naturally occurring compounds isolated from bacteria, but they exhibited poor antibacterial activity. These naturally occurring monobactams are characterized by the 2-oxoazetidine-1-sulfonic acid moiety with an acyl side chain at the 3 position with the β-orientation and, for most monobactams, a 3-α-methoxy group. The potent antibacterial activity of aztreonam is directed specifically against aerobic gram-negative bacteria. This activity varies from high against organisms such as Neisseria and Haemophilus spp. to intermediate against Pseudomonas aeruginosa to poor against Acinetobacter spp.
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Chapter 11 : β-Lactam Compounds as β-Lactamase Inhibitors
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An understanding of the mechanism of action and the inactivation of native and mutant β-lactamases, together with the identification of positions where amino acid substitutions occurred and the folding properties and flexibility of these enzymes, is important for the design of new effective β-lactam antibiotics as well as inhibitors of these enzymes. Scientists have identified different processes for the inactivation of Escherichia coli TEM-1 (RTEM) β-lactamase by sulbactam. First, sulbactam is a substrate in the sense that the enzyme catalyzes the hydrolytic opening of the β-lactam ring. Second, it is an enzyme inhibitor; at pH 8, about 10 molecules of inhibitor are consumed per enzyme molecule. Thus, interaction of the enzyme with sulbactam gives rise to irreversible inhibition. Clavulanic acid, sulbactam, and tazobactam inhibit exocellular β-lactamases encoded by plasmids from Staphylococcus spp. and group 2 periplasmic β-lactamases (except for some TEM mutants) of gram-negative bacteria. These β-lactamases include the TEM-1 β-lactamase, which is plasmid encoded and is one of the most common β-lactamases found in bacteria. Numerous studies have demonstrated the effectiveness of the clavulanate mixture in primary and recurrent urinary infections caused by susceptible strains of E. coli and other bacteria, including many amoxicillin-resistant strains. It has been used with good results in infections of the skin and soft tissues caused by susceptible pathogens, including β-lactamase-producing strains of Staphylococcus aureus. The broad antibacterial spectrum of piperacillin-tazobactam mixture makes it most useful for the treatment of infections in immunodepressed or neutropenic patients.
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Chapter 12 : Bacterial Resistance to β-Lactam Antibiotics and β-Lactam Inhibitors of β-Lactamases
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The expanding problem of resistance to β-lactam antibiotics and β-lactam inhibitors of β-lactamases illustrates the genetic adaptability of bacterial populations. The evolution of bacterial resistance to the action of these drugs occurred over a relatively short period. Bacteria become resistant to antibiotics and β-lactam compounds acting as β-lactamase inhibitors either through mutations or by acquisition of specific resistance genes from other bacteria. Global antibacterial resistance is an increasing public health problem. The pharmaceutical industries are reacting to the problem by discovering novel antibacterial agents to overcome the emergence of bacterial resistance to antibiotics and β-lactamase inhibitors. A section of the chapter summarizes the status of the development of structural modifications of existing groups and subgroups of antibacterial agents to make them less susceptible to degradation by β-lactamases, to increase penetrability through the outer membrane of gram-negative bacteria, or to have an increased affinity for mutated penicillin-binding proteins (PBPs). The search for new anti-methicillin-resistant Staphylococcus aureus (MRSA)-lactam antibiotics appears to be focused on developing agents that inhibit PBP2a, which gives rise to methicillin resistance in staphylococci and penicillin-resistant pneumococci. The increase in β-lactam antibiotic resistance due to the production and rapid spread of resistance encoded by plasmids or transposons in pathogenic bacteria has made the enzymes an attractive target for drug development.
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Chapter 13 : Inhibitors of Peptidoglycan Biosynthesis
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The structure and biosynthesis of the peptidoglycan unit have special significance relative to the action of the antibiotics fosfomycin, D-cycloserine, and bacitracin and the glycopeptides vancomycin and teicoplanin, as well as the β-lactam antibiotics. The biosynthesis of peptidoglycan was first worked out with Staphylococcus aureus. Although bacteria show variations in peptidoglycan structure, the biosynthetic sequence in S. aureus serves to illustrate the general features of the process. The biosynthesis of peptidoglycan may be conveniently divided into stages inhibited by fosfomycin and those inhibited by D-cycloserine antibiotics. Fosfomycin acts as a phosphoenolpyruvate analogue and irreversibly inhibits the enol-pyruvyl transferase that catalyzes the transfer of phosphoenolpyruvate in the formation of UDP–N-acetylglucosamine (UDPNAG) enolpyruvate. In multiple-dose regimens, resistance to fosfomycin emerges rapidly; however, cross-resistance with other antibacterial agents has not been common. Resistance to fosfomycin occurs by three mechanisms; two of them are encoded on the chromosome, whereas the third is of plasmid origin. Fosfomycin is inactivated by the opening of its epoxide ring followed by formation of an adduct between its C-1 atom and the sulfhydryl group of the cysteine of the tripeptide glutathione. D-Cycloserine is now used only for the treatment of patients with tuberculosis whose Mycobacterium tuberculosis strains are resistant to several first-line drugs. Resistance in Mycobacterium tuberculosis is rare and develops only slowly in patients treated with D-cycloserine alone.
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Chapter 14 : Inhibitors of Peptidoglycan Biosynthesis
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The action of vancomycin and teicoplanin depends on their ability to bind specifically to the terminal D-alanyl-D-alanine group on the peptide side chain of the membrane-bound intermediates in peptidoglycan synthesis. Bacitracin was isolated in 1943 from a strain of a Bacillus sp., which was originally classified as Bacillus subtilis but now is known as Bacillus licheniformis. Binding of bacitracin prevents the enzymatic dephosphorylation of the lipid carrier molecule to its monophosphate form, a reaction which occurs during the second stage of peptidoglycan biosynthesis. Bacitracin is highly active against most gram-positive bacteria, particularly Staphylococcus aureus and Streptococcus pyogenes. Vancomycin binds reversibly to the D-Ala–D-Ala dipeptide segment of the muramyl pentapeptide present in peptidoglycan monomers which are exposed at the external cell surface of the cytoplasmic membrane. The dimeric structure of vancomycin is held together by four hydrogen bonds between the two amide backbones. Dimerization results in an enhanced antibacterial activity of vancomycin and other glycopeptides through cooperative binding effects. Vancomycin is the antibiotic of choice for serious infections caused by methicillinresistant S. aureus (MRSA) and coagulase-negative staphylococci, including methicillinresistant S. epidermidis. The development of glycopeptide antibiotics with activity against vancomycin- and teicoplanin-resistant organisms is of utmost importance because of the recent emergence of low-level vancomycin resistance in S. aureus and the prevelance of vancomycin-resistant enterococci (VRE) in immunocompromised patients.
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Chapter 15 : Antibiotics That Affect Membrane Permeability
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The polymyxins are a group of cyclic, polycationic peptide antibiotics with a fatty acid chain attached to the peptide through an amide linkage. They are produced by fermentation of strains of Bacillus polymyxa. Polymyxins B and E (colistin) are the least toxic and are the only polymyxins used clinically. These antibiotics contain a 7-amino-acid ring attached to a 3-amino-acid tail, to which is attached a fatty acyl group. It has been suggested that the fatty acid part of the polymyxin molecule penetrates into the hydrophobic region of the outer membrane and the ammonium groups interact with the lipopolysaccharides and phospholipids, competitively displacing divalent cations (calcium and magnesium) from the negatively charged phospholipid group of the membrane lipids. This displacement disrupts membrane organization and increases the permeability of the membrane. Polymyxins B and E are active almost exclusively against aerobic gram negative bacilli. In particular, they exhibit quite good activity against Pseudomonas aeruginosa. In the past, these polymyxins were often used for the treatment of P. aeruginosa infections. Nowadays, because of the availability of effective and less toxic drugs such as gentamicin, tobramycin, amikacin, ticarcillin, piperacillin, and ceftazidime, polymyxins are not the antibiotics of choice to treat infections caused by this bacterium.
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Chapter 16 : Antibiotic Inhibitors of Bacterial Protein Synthesis
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This chapter gives an overview of the structure of tRNA to provide a useful outline for understanding how tRNAs serve as interpreters in the translation of mRNA nucleotide sequences into the amino acid sequence of a polypeptide chain. First, methionine is attached to tRNAf Met by the Met-tRNA synthetase. The initiation complex is now ready to initiate the elongation step. Protein synthesis stops when the ribosome reaches one of the three special nonsense codons—UAA, UAG, and UGA. Although the mechanism of protein synthesis is similar in prokaryotes and eukaryotes, prokaryotic ribosomes differ substantially from those in eukaryotes. Studies of protein folding have led to the observation that large protein chains use molecular chaperones to adopt their correct conformation. The puromycin-peptide complex is then released from the ribosome, halting the elongation step.
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Chapter 17 : Inhibitors of the 30S Ribosomal Subunit
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The aminoglycoside group of antibiotics are multifunctional hydrophilic carbohydrates that possess two or more amino monosaccharides connected by glycosidic bonds to an aminocyclitol nucleus. Most aminoglycosides contain a 2-deoxystreptamine cyclitol. The aminoglycoside antibiotics have several features justifying their continued clinical use, including their rapid and potent bactericidal activity, long-lasting postantibiotic effect, and synergy with other antibiotics. The emerging structural data now have the potential to be exploited in the design of specific inhibitors of enzyme activity. The challenge is to use this information to synthesize effective and potent inhibitors that will overcome antibiotic resistance produced by the aminoglycoside-modifying enzymes. The tetracyclines are a group of antibiotics with an identical basic skeleton of four linearly fused six-membered rings, named 1,4,4a,5,5a,6,11,12-octahydronaphthacene and differing from each other chemically only by substituent variation at positions 5, 6, and 7. It has been found that there are two binding sites for tetracycline within the small ribosomal subunit. Bacterial resistance results from the selective pressure exerted on bacteria during the administration of tetracyclines for chemotherapy. Resistance to tetracycline may be mediated by one of three different mechanisms: (i) an energy-dependent efflux of tetracyclines carried out by transmembrane spanning proteins, which results in reduction of the concentration of tetracycline in the cytosol; (ii) ribosomal protection, whereby the tetracyclines no longer bind productively to the bacterial ribosome; or (iii) chemical modification, requiring oxygen and NADPH and catalysis by enzymes. Since the chemical alteration mechanism occurs rarely, this discussion focuses on the major mechanisms of tetracycline resistance.
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Chapter 18 : Inhibitors of the 50S Ribosomal Subunit
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The macrolides are classified according to the number of atoms comprising the lactone ring in 12-, 14-, 15-, or 16-member macrolides, each of which has both chemical and biological differentiating characteristics. Target site modification has resulted in cross-resistance to the macrolides, lincosamides, and streptogramin B, the so-called MLSB phenotype. The natural macrolide antibiotics are isolated primarily from the genus Streptomyces. They are characterized by having antibacterial activity mostly against gram-positive bacteria. The macrolide antibiotics currently used in the United States are erythromycin and the semisynthetic derivatives of erythromycin A, i.e., clarithromycin, azithromycin, and dirithromycin. Constitutive resistance occurs when the methylating enzyme is produced constitutively, and inducible resistance occurs when the enzyme induction is effected by exposure of the organism to both 14-member ring and 15-member ring but not 16-member ring macrolides. The investigators conclude that the differences between the binding modes of macrolides, azalides, and ketolides reveal the contributions of the specific chemical modifications of the macrolides, and they explain the enhanced binding properties of the advanced compounds on this basis. The mode of action of the group B streptogramins is thought to be similar to that of 14-member ring macrolides, which sterically hinder the growth of the nascent peptide during early rounds of translation. Resistance to class B streptogramins takes place (i) by rRNA methylation catalyzed by a 23S rRNA methylase encoded in the erm genes, which also confers resistance to macrolides and lincosamides, or (ii) by an elimination of the hexadepsipeptide ring by specific lyases.
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Chapter 19 : An Inhibitor of the 50S Ribosomal Subunit
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Chloramphenicol was the first orally active broad-spectrum antibiotic to be discovered. A valuable property of chloramphenicol is that it readily crosses the blood-brain barrier and can therefore be used to treat infections of the central nervous system caused by susceptible organisms. The aplasia is not dose related and can become manifest weeks to months after the use of chloramphenicol. Chloramphenicol is therefore reserved for situations where the benefits exceed the risk. Chloramphenicol is known to bind at the peptidyltransferase center of the large ribosomal subunit. To elucidate the structural basis of ribosome-chloramphenicol interactions, scientists have determined the high-resolution X-ray structure of the 50S ribosomal subunit of the eubacterium Deinococcus radiodurans in complex with chloramphenicol. The most clinically important mechanism of resistance in bacteria is that of O acetylation catalyzed by the enzyme chloramphenicol O-acetyltransferase (CAT). This chapter discusses the postulated general mechanism for the CAT-catalyzed acetylation of chloramphenicol. Although the CAT mechanism for resistance to chloramphenicol is widespread in bacteria, it is not used by the chloramphenicol-producing Streptomyces strains to protect themselves against their own toxic product. However, a 3-O phosphoester of chloramphenicol was identified in Streptomyces venezuelae, suggesting that the producing organism has a mechanism of chloramphenicol resistance that has not been encountered in other microbial systems. Researchers reported their studies on active efflux of chloramphenicol in susceptible E. coli strains and in multiple-antibiotic-resistant (Mar) mutants; the mechanism was shown to depend on proton motive force.
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Chapter 20 : Inhibitors of the Formation of the First Peptide Bond
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The oxazolidinones are a novel chemical class of synthetic antibacterial agents that target protein synthesis in a wide spectrum of gram-positive pathogens including methicillin-resistant Staphylococcus aureus (MRSA), penicillin-resistant Streptococcus pneumoniae, and vancomycin-resistant Enterococus faecium. In 1987 the antibacterial activities of the oxazolidinones were first described by scientists at E. I. du Pont de Nemours & Co., Inc. It was demonstrated that the oxazolidinone DuP721 inhibited protein synthesis in susceptible bacteria. Oxazolidinones were abandoned for some time after these earlier studies because of their high toxicity. Oxazolidinones are potent inhibitors of bacterial protein biosynthesis. Several hypotheses regarding the mode of inhibition of protein synthesis in sensitive bacteria by oxazolidinone have been proposed. Scientists found that these oxazolidinones inhibit ribosomal peptidyltransferase activity in the simple reaction of 70S ribosomes. Then they proposed that oxazolidinones inhibit bacterial protein biosynthesis by interfering with the binding of initiation fMet-tRNA to the ribosomal peptidyltransferase P site, which is vacant only prior to the formation of the first peptide bond. Then oxazolidinones do not affect the formation of the 30S preinitiation complex but do prevent the formation of the fMet-tRNA–ribosome– mRNA ternary complex. In 2002, Auckland and coworkers reported the first three examples of resistant enterococci (two isolates of E. faecium and one of E. faecalis) isolated in the United Kingdom; they were obtained from patients who had received linezolid.
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Chapter 21 : Inhibitor of Isoleucyl-tRNA Synthetase
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Mupirocin was isolated as the major component of a family of structurally related antibiotics produced by a strain of Pseudomonas fluorescens. The chemical structure of mupirocin, 9-[2(E)4-(2S,3R,4R,5S)[5-(2,3-epoxy-5-hydroxy-4-methylhexyl)-3,4-dihydroxytetrahydropyran-2yl]-3-methylbut-2-enoyloxy]nonanoic acid, was established based on nuclear magnetic resonance spectroscopy, mass spectrometry, and chemical degradative studies of its methyl ester and various derivatives. Mupirocin inhibits the growth of staphylococci (including methicillin-resistant strains) and streptococci (except enterococci) at low concentrations and is bactericidal at high concentrations, which are readily achieved by topical application. Mupirocin has no cross-resistance to existing antibacterial agents, is most active at acidic pHs against bacteria included in its spectrum, and penetrates well into superficial layers of the skin and nasal mucosa. Mupirocin has been successfully used in eradication of the nasal carrier state of Staphylococcus aureus, including methicillin-resistant S. aureus (MRSA). Patients with folliculitis, furunculosis, impetigo, or other primary skin infections have been treated successfully with mupirocin. Mupirocin is an analogue of isoleucine; it competitively binds Isoleucyl-tRNA synthetase (IleRS) joins isoleucine to tRNA at its synthetically active site. The 2.2Å-resolution crystal structure of S. aureus IleRS complexed with tRNAIle and mupirocin has recently been solved. Mupirocin resistance in S. aureus results from changes in the target enzyme, IleRS. These resistant strains can be divided arbitrarily into two distinct groups, those which exhibit a low level of resistance and those which exhibit a high level of resistance.
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Chapter 22 : Inhibitors of DNA-Dependent RNA Polymerase
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The clinically useful rifamycins include rifampin (known in some countries as rifampicin), a semisynthetic derivative of rifamycin B. Rifamycin B and structurally related antibiotics inhibit bacterial RNA synthesis by binding to the β-subunit of bacterial DNA-dependent RNA polymerases. A section of the chapter provides an overview of transcription in bacteria and the structure and function of bacterial DNA-dependent RNA polymerase. In the initiation phase of RNA synthesis on a duplex DNA template, the RNA polymerase binds at a specific site on the DNA called the promoter. Here, it unwinds and unpairs a small segment of DNA. Rifamycins are characterized by an aliphatic ansa-bridge that connects two nonadjacent positions of a naphthalenic nucleus. Rifamycin SV can be obtained by chemical modification of other rifamycins but is also produced by fermentation of some strains of A. mediterranei. The rifamycins are biologically active against gram-positive bacteria and mycobacteria, particularly Mycobacterium tuberculosis, the agent of tuberculosis. The mechanism of action of rifabutin against M. avium is unknown; in some other susceptible bacteria, this antibiotic inhibits DNA-dependent RNA polymerase. Rifapentine, like other rifamycins, inhibits DNA-dependent RNA polymerase. In addition to its activity against M. tuberculosis, it is active in vitro against M. avium and Toxoplasma gondii. Strains of M. tuberculosis resistant to rifamycin are also resistant to rifapentine. A major advantage of rifapentine over rifampin is that only twice-weekly doses of rifapentine are required for initial treatment of tuberculosis and once-weekly doses are needed during the continuation phase of treatment.
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Chapter 23 : Inhibitors of DNA Gyrase and Topoisomerase IV
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DNA gyrase and topoisomerase IV cleave double stranded DNA in both strands and then transport another segment of double-stranded DNA through the cleaved DNA segment before religating the DNA. Topoisomerases are divided into two classes, type I and type II. Type I topoisomerases, represented by Escherichia coli topoisomerases I and III, break single strands of duplex DNA, pass another single DNA strand through the break, and then reseal the break. In contrast, type II topoisomerases, represented by E. coli DNA gyrase and topoisomerase IV, break both strands of duplex DNA, pass other DNA duplex strands through the break, and reseal both breaks. Bacterial resistance to quinolones may result from chromosomal mutations coding for modifications in target subunits of bacterial topoisomerases II and IV or by active efflux via efflux pumps and alterations in the expression of outer membrane proteins, most importantly OmpF. The quinolones most strongly affected by a single mutation are those for which the mutations occur in their preferred target, for example in gyrase for sparfloxacin, grepafloxacin, gatifloxacin, gemifloxacin, clinafloxacin, and moxifloxacin and in topoisomerase IV for ciprofloxacin, norfloxacin, levofloxacin, and trovafloxacin. Resistance mutations in GyrB generally occur less often than those in GyrA and have been found in E. coli, Salmonella enterica serovar Typhimurium, Mycobacterium tuberculosis, Staphylococcus aureus, and Streptococcus pneumoniae. Quinolones must traverse the cell wall and cytoplasmic membrane of gram-positive bacteria and, additionally, the outer membrane of gram-negative bacteria to reach the DNA gyrase and topoisomerase IV in the cytoplasm.
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Chapter 24 : Antibacterial Agents That Cause DNA Damage in Obligate Anaerobic Organisms
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This chapter focuses on metronidazole that has become an extremely important antibacterial agent, especially in the treatment of anaerobic bacterial infections. It causes bacterial DNA damage regardless of the growth phase of the organism and is rapidly bactericidal. The observation that metronidazole relieved acute ulcerative gingivitis in a patient being treated for trichomonal vaginitis led to studies, culminating in 1962, of its use in anaerobic bacterial infections. Subsequently, it was confirmed that metronidazole was useful for the treatment of Vincent's stomatitis and that it inhibited Fusobacterium necrophorum. According to published data, the selective activity of 5-nitroimidazoles (metronidazole and tinidazole) against anaerobic organisms is due to the preferential reduction of the 5-nitro group by obligate anaerobes but not by aerobes. Understanding of the antimicrobial resistance to metronidazole is based on studies with anaerobic microorganisms such as Bacteroides, Trichomonas, and Clostridium spp. Although resistance rates of Trichomonas vaginalis are low, treatment failures due to resistance are significant. The MIC of metronidazole for T. vaginalis causing refractory vaginitis is frequently three to eight times the MIC for susceptible strains.
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Chapter 25 : Antibacterial Agents That Cause Damage to DNA
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The 5-nitrofurans are a class of totally synthetic antibacterial agents characterized by being derivatives of 5-nitrofuran and by containing the azomethine group (--CH=N--). Nitrofurantoin is still in use in humans because of its broad spectrum of activity, which covers gram-positive and gram-negative bacteria (except Pseudomonas aeruginosa and some Klebsiella and Proteus strains), its relatively low toxicity, and the infrequent development of resistance. Since the introduction of nitrofurantoin for use in urinary tract infections (UTIs), essentially no resistance has occurred, unlike other antibacterial agents. It was suggested that this is due to the ability of the 5-nitrofurans to affect multiple cytoplasmic targets such as inhibiting various enzymes within bacteria and that they may also damage bacterial DNA, leading to DNA strand breakage. Urinary antiseptics are antibacterial agents that concentrate in the urine but do not produce adequate levels in serum. Therefore, these antibacterial agents are useful only in the prevention or therapy of lower UTIs and not for treatment of severe pyelonephritis or associated systemic infections. Bacteria have no mitochondria; in bacteria, the mitochondrial functions are all carried out within the plasma membrane, where all the needed enzymes are anchored. This relatively poor protection of the enzyme system (compared with that in the host cell) could explain the selective toxicity of these 5-nitro aromatic antibacterial agents.
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Chapter 26 : Compounds That Interfere with Tetrahydrofolic Acid Biosynthesis
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Two groups of compounds, the sulfonamides and the 2,4-diaminopyrimidines, e.g., trimethoprim (TMP), interfere with the biosynthesis of tetrahydrofolic acid (THF). Their chemistry and development are treated separately, but their mechanism of action relies on the inhibition of two different enzymes, dihydropteroate synthetase (DHPS) and dihydrofolate reductase (DHFR), which are active in the bacterial biosynthesis of THF. Sulfonamides derived from p-aminobenzenesulfonamide are commonly referred to as sulfa drugs. Sulfonamides are synthetic antibacterial agents with an illustrious history. Chemically, the clinically useful sulfonamides are derived from paminobenzenesulfonamide. The enzyme DHPS catalyzes the displacement of pyrophosphate from the pteridine substrate. It is well established that the sulfonamides mimic p-aminobenzoic acid (PABA), which is the natural substrate required for the biosynthesis of THF. Since it does not enter bacterial cells, bacteria must synthesize dihydrofolic acid (DHF) and THF intracellularly de novo. This difference in the biochemistry of the bacterial cell and the human cell is the basis of the selective toxicity of the sulfonamides. Huovinen indicated that it may be difficult to separate the influences of the transport-related mechanisms on resistance levels. P. aeruginosa has these types of resistance mechanisms, which explains why the potency of TMP and the sulfonamides against P. aeruginosa is limited, with MICs typically in the resistant range. For certain pathogens, TMP-Sulfamethoxazole (SMX) is considered the agent of choice. These include Moraxella catarrhalis, Haemophilus influenzae causing upper respiratory infections and bronchitis, Y. enterocolitica, Aeromonas spp., Burkholderia cepacia, S. maltophilia, and Nocardia spp.
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Chapter 27 : New Antibacterial Drugs in Development That Act on Novel Targets
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This chapter presents a survey of some new antibacterial agents that act on novel targets. In recent years, bacterial resistance to antibacterial drugs has become a global public health threat and has been increasing due to the use, overuse, and misuse of broad-spectrum antibiotics and the ability of bacteria to exchange resistance genes. Cationic peptides exhibit a broad spectrum of activity against various targets, including gram-negative and gram-positive bacteria, fungi, enveloped viruses, and parasites. Aminoacyl-tRNA synthetases play a crucial role in protein synthesis in all organisms, and selective inhibition of the bacterial enzymes has potential for the discovery of new antibacterial agents. Uropathogenic strains of Escherichia coli are the primary causative agents of urinary tract infections in humans. Combinatorial chemistry has had a significant impact on the discovery of new antibacterial drugs. Most of the successes have come from the use of small libraries to explore a specific pharmacophore. This kind of application has been exemplified in the chapter with the discovery of actinonin, a selective peptide deformylase inhibitor. The traditional method for obtaining new antibacterial drugs has been to synthesize analogues of existing antibacterial drugs and evaluate them for improved therapeutic activity by using in vitro and in vivo methods that detect antibacterial activity against gram-positive and gram-negative organisms.
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