Bacteria versus Antibacterial Agents: an Integrated Approach

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.