Antibiotics: Actions, Origins, Resistance

Major antibacterial drugs in current human use can be categorized in multiple ways. One is by economic impact and the other is by the bacterial diseases they are prescribed to treat. While the antitubercular drug rifampin and the carbapenem version of the β-lactam imipenem are listed in this chapter, representative brand names are also indicated, as well as infections for which these drugs have been utilized and where clinically significant resistance had been detected. These classes of antibiotics are taken up in detail in the chapter, with discussions of mechanisms of action, modes of resistance development, and prospects for development of new versions to overcome resistance. The gram-positive Mycobacterium tuberculosis still causes millions of deaths annually. The historical scourges of plague and cholera are caused by two gram-negative bacteria, Yersinia pestis and Vibrio cholerae, respectively, while E. coli, Salmonella and Shigella strains are common causes of diarrheal diseases. The gram-negative Pseudomonas aeruginosa is often described as an opportunistic pathogen, causing disease in settings where the patient may have compromised immunity. The mechanisms of action of most antibacterial drugs were worked out after the discovery that the molecules had effects on bacterial growth, either slowing growth dramatically (bacteriostatic) or killing the bacteria (bactericidal). The cell wall biosynthetic processes and protein biosynthesis on the ribosome historically have been the site of action of the largest number of antibiotics, perhaps because of the many enzymatic steps, which offer multiple opportunities for disrupting key attributes of a healthy bacterial cell.

This chapter deals with antibiotics that interdict any of the several steps in bacterial cell wall assembly, from biogenesis of the dedicated monomers to the specialized assembly, membrane translocation, and extracellular cross-linking and strengthening of the exoskeletal peptidoglycan (PG) layers. Many of the antibiotics that affect bacterial cell walls inhibit enzymes or sequester substrates involved in peptidoglycan assembly and cross-linking. Distinct features of outer membranes even among gram-negative bacteria can lead to differences in permeability to antibiotics. For example, Pseudomonas aeruginosa outer membranes show about 100-fold lower permeability to cephalosporins such as cephaloridine than other gram-negative bacteria, in part because of porins with small pores to reduce inward passage of the antibiotics into the periplasmic space. Some of the transglycosylases and transpeptidases are bifunctional with discrete transglycosylase and transpeptidase domains, and members of this subset are of particular importance as killing targets of β-lactam antibiotics. The most celebrated of the antibiotics that kill bacteria by blocking the crucial transpeptidations that lead to mechanically strong PG through the covalent cross-links of peptide strands are the β-lactam antibiotics. Moenomycin has a 25-carbon lipid alcohol, moecinol, linked via a phosphoglycerate to a pentasaccharide tail in phosphodiester linkage. NMR analysis has provided a model for the three-dimensional structure with the proposal that the E and F rings of the carbohydrate moiety interact, as a substrate analog, with the target transglycosylase to shut down addition of the disaccharyl pentapeptide units in PG layer growth.

This chapter deals with the various classes of antibiotics that exert their bacteriostatic or bactericidal action by blockade of one or more of the protein biosynthetic steps that occur on the 30S and 50S subunits of the bacterial ribosome. It presents a summary on the ribosome and then analyzes the sites and mechanism of action of ribosome-inhibiting antibiotics. The peptidyl chain is translocated onto the aminoacyl-tRNA in the A site by the peptidyltransferase activity in each peptide-chain-elongation cycle of the ribosome. Architectural differences in the 23S RNA of bacterial ribosomes versus their eukaryotic counterparts provide selectivity for killing of the bacteria. The recent X-ray analysis of macrolide antibiotics bound to bacterial ribosomes gives some insight into this selectivity. The determination of the structure of the 30S ribosomal subunit from Thermus thermophilus with bound drug has revealed a major binding site and a lower-affinity binding site for tetracycline. Aminoglycosides are potent drugs against gram-negative bacteria but not very effective against gram-positive organisms, although the combination of aminoglycosides and β-lactams is used to treat enterococcal infections. It has been reported that linezolid-resistant mutants map to the 23S rRNA sites near the peptidyltransferase center, consistent with recent kinetic studies showing that oxazolidinones are competitive inhibitors of both A-site and P-site substrates.

Inhibition of DNA replication and repair enzymes would seem a logical target for antibacterial action by natural products elaborated by microbes to kill their neighbors. One such class of molecules, the coumarins, represented by such streptomycete metabolites as novobiocin and coumermycin, has been studied for many years and served to pinpoint enzymes called DNA type II topoisomerases, specifically DNA gyrase, as the killing target. The newest generation of quinolones in particular, such as gatifloxacin, have increased potency against gram-positive pathogens. Thousands of fluoroquinolones have been synthesized around the core planar heterocyclic nucleus that gives the family its name. Extensive analysis has indicated that quinolones affect the double-strand cleavage/double-strand religation equilibrium in gyrase and topo IV catalytic cycles, such that the cleaved complex accumulates. There has been speculation about whether quinolones speed up the double-cleavage step of bound DNA or selectively slow the double-religation step, without definitive evidence for either interpretation. The mechanism by which quinolones induce the accumulation of the doubly cut covalent DNA-enzyme intermediate is likewise still mysterious. As the quinolone-covalent gyrase-doubly cut DNA intermediate accumulates, the killing action is thought to be from the downstream effect this block has on the progression of DNA replication forks which are halted by this. It may be that DNA repair machinery is recruited, attempts to come to the rescue, and fails as the recalcitrant quinolone-stabilized gyrase-DNA intermediate persists. This may be the signal that turns on the signaling processes that lead to the rapid killing of bacteria induced by the quinolones.

The sulfa drugs are considered as the longest used class of synthetic chemicals. These drugs were first tested in the 1930s as bacteria-killing molecules. The current generation of sulfa drug is sulfamethoxazole, used in combination with trimethoprim for the treatment of patients with urinary tract infections and also for AIDS patients with Pneumocystis carinii infections. This drug pair also validates that combination chemotherapy can be an effective strategy in curing bacterial infections. Each of the drug molecules blocks a step in folic acid metabolism. Thus, the rationale for the combination is synergistic blockade of two different steps in the biochemistry of this essential coenzyme. A set of peptides with antibiotic activity are produced by gram-positive bacteria and are classified as lantibiotics because they all contain the unusual double-headed thioether-containing amino acid lanthionine or its β-methyl lanthionine congener. Rifampin is used clinically only as part of combination regimens for killing the slow-growing pathogen Mycobacterium tuberculosis. The drug is an RNA polymerase inhibitor, the only one in clinical use for blocking bacterial transcription. Rifampin binds to the β subunit of the RNA polymerase enzyme at an allosteric site, not at the active site, as defined by resistant mutations in clinical isolates of M. tuberculosis and M. leprae. Clinical isolates of rifamycin-resistant M. tuberculosis have mutations in the β subunit residues that recognize rifamycin, with about three-quarters of the resistance arising from mutations in side chains of residues 406 and 411.

Plasmid sized DNA elements can integrate into specific attachment sites on chromosomes to create antibiotic resistance islands, as found in Salmonella enterica serovar Typhimurium DT104 and methicillin-resistant Staphylococcus aureus (MRSA). This allows multiple resistance genes to be maintained together. All these routes ensure rapid spread and stable maintenance of collections of antibiotic resistance genes through bacterial populations. Many antibiotics such as mitomycin and vancomycin have been developed into approved antibacterial drugs and have MICs or sometimes 50% inhibitory concentrations, often in Petri plate assays, in the range of 1 µg/ml. Many observers have noted that antibiotic producers could be vulnerable to their own chemical weapons of destruction and must have worked out strategies for their own protection and immunity. Three strategies for self-resistance have been described in macrolide producers and presage acquired resistance mechanisms in human pathogens. This chapter provides examples that typify the kinds of acquired resistance mechanisms that have presumably been accumulated, some from the reservoir of these genes in producer organisms and some from evolution of housekeeping enzymes to new specificities, by soil bacteria in the hundreds of millions of years that they have coevolved with antibiotic-producing neighbors. The three methods of self-protection in macrolide antibiotic producers set the stage for the understanding of the three major strategies for bacterial resistance to antibiotics.

Enzymatic inactivation of antibiotics occurs with several of the natural product antibiotic classes but has not yet been observed as a major route of resistance development for the classes of synthetic antibacterials: the sulfamethoxazole-trimethoprim combination, the fluoroquinolones, or the oxazolidinones. The most widespread mode of clinical resistance development to β-lactam antibiotics is the expression of β-lactamases that hydrolyze the antibiotic. Two approaches have been taken in the decades since lactam-resistant clinical isolates began to diminish the efficacy of penicillins and cephalosporins as antibiotics. The first has been to develop semisynthetic β-lactams which were slower substrates for attack by the hydrolytic lactamases. The second approach has been to screen for inhibitors and inactivators of lactamase activity and then combine these molecules with a β-lactam. β-Lactamase genes can be embedded in bacterial chromosomes, such as the ampC gene in enteric bacteria or the blaZ gene in Staphylococcus aureus, or they can be carried on multiple-copy plasmids or transposons, as is the case for the TEM-1 bla gene in a variety of high-level penicillin-resistant gram-negative bacteria found in clinical isolates. In Escherichia coli the ampG, ampD, and ampR genes control expression of the ampC-encoding β-lactamase. In S. pneumoniae external penicillin leads to an increase in autolytic peptidoglycan hydrolase activity and subsequent vulnerability to osmotic lysis and death. Three kinds of enzymatic modifications of OH and NH2 groups on aminoglycosides are common determinants of resistance and represent variants of normal electrophilic group transfer enzymes that participate in primary metabolism.

Active efflux can be clinically relevant for β-lactam antibiotics, macrolides, the pristinamycin peptides, fluoroquinolones, and most classically the tetracyclines. From bioinformatic analysis four protein families of efflux pumps that can function in antibiotic resistance have been described. The pumps driven by proton motive force (ΔpH) are categorized in the major facilitator subfamily (MFS), the small multidrug regulator (SMR) family, or the RND (resistance/nodulation/ cell division) family, based on projected size and the need for partner proteins and subunits. A significant breakthrough in understanding the architecture of an ABC type transporter has been obtained by crystallization of the MsbA protein from Escherichia coli, at a relatively low resolution, but sufficient to reveal orientation of nucleotide binding domains (NBDs) to transmembrane domains (TMDs) and allowing a model for transporter action. MsbA is homologous to human MDR-1 and mouse MDR3, multidrug resistance transporters that are thought to act physiologically as lipid and phospholipid ‘‘flippases,’’ moving phospholipid molecules from the inner to the outer layer of the membrane bilayer. E. coli O157:H7 exhibits resistance to streptomycin, tetracycline, and sulfa drugs and may do so through reduction of outer membrane permeability for uptake.

One of the routes to clinically important resistance in pathogenic bacteria is the ability of drug-resistant pathogens to modify the drug target to insensitivity while still retaining its essential cellular function. This chapter exemplifies the principles of antibiotic resistance arising from replacement or modification of the target. This can be achieved by mutation at one or more sites in the target gene or by importation of a gene that specifies a new replacement enzyme that has markedly decreased sensitivity to the drug. β-lactam resistance in the grampositive Streptococcus pneumoniae and Staphylococcus aureus strains represent these two variations on a theme. Unlike the S. aureus strains and many other pathogens, S. pneumoniae does not use β-lactamases as the major route to penicillin resistance. Analysis of transpeptidases/transglycosylases in S. pneumoniae reveal five high-molecular-weight PBPs which contribute to killing by β-lactams. One of the goals of medicinal chemistry in developing broad-spectrum erythromycin family of macrolides is to overcome the Erm phenotypes by creating semisynthetic or altered versions of the macrolides that can still bind to methylated A2058 versions of the 23S rRNA. Telithromycin has recently been approved for human use and ABT-773 is in advanced clinical evaluation. Enterococcus faecalis species account for about 90 to 95% of vancomycin-resistant clinical isolates and E. faecium another 5%, with minor species accounting for the rest. There had been few therapeutic choices for vancomycin-resistant enterococci (VRE) treatment, but the recent approvals of both the Synercid combination and the oxazolidinone linezolid indicate efficacy against VRE.

Of the circa 12,000 known antibiotics, it has been estimated that some 160 are or have been in human clinical use. Streptomycetes, gram-positive filamentous bacteria, account for these production of about 55% of the commercially significant antibiotics. The Abs knockout leads to precocious production of all the Streptomyces coelicolor antibiotics in hours to days and in amounts up to 60-fold higher than normal, dependent on the culture conditions. The most extensive analysis of regulation of a specific antibiotic pathway, one step down from the Abs global regulators, is probably in Streptomyces virginiae in production of the two antibiotics virginiamycin M1 and virginiamycin S1. The hydrophobic side chain of the butyrolactones, known generically also as butaneolides, is in the same locus as the N-acyl moiety of the gram-negative quorum molecules. A schematic for regulation of biosynthetic gene expression for the major classes of streptomycete antibiotics (polyketides, nonribosomal peptides, and aminoglycosides) is beginning to take shape. Plant pathogenic bacteria often secrete enzymes (exoenzymes) with hydrolytic capacity to destroy the components of plant cell walls to release the nutrients that can then be utilized by the pathogens. The N-acylhomoserine lactones of Erwinia, Pseudomonas, and many other bacteria and the γ-butyrolactones of streptomycetes, serve equivalent purposes, as low-molecular-weight pheromones, for communicating population density-dependent signals between bacteria of the same species.

The chemistry practiced by polyketide synthases (PKSs) is closely parallel to that of fatty acid synthases (FASs), which have been extensively studied for decades to decipher mechanisms and organization and have provided precedents for understanding PKS logic. The four rings of the tetracycline family of antibiotics, such as oxytetracycline and chlortetracycline and also of tetracenomycin and the antitumor antibiotic doxorubicin are produced by type II polyketide synthases along with some partner aromatases and cyclases also expressed in the clusters. In biosynthesis of daunorubicin or doxorubicin, the starter unit is propionyl-CoA and all extender units are malonyl-CoA. In biosynthesis of daunorubicin or doxorubicin, the starter unit is propionyl-CoA and all extender units are malonyl-CoA. After nine condensation cycles a decaketidyl-S-ACP is built up, in which all 10 of the ketone groups are thought to persist without reductive modification in the acyl chain. The first cyclase, TcmN, closes three rings to make TcmF2, and TcmI closes the last ring to give the fused four-ring system analogous to the tetracycline skeleton. The macrolides of the erythromycin class contain 14-membered macrolactone rings while the tylosins have 16-membered rings. Many of the macrolide antibiotics are glycosylated, to provide hydrogen bond interactions with the ribosome 23S rRNA, highlighted by the contacts of the desosamine sugar of erythromycin to A2058. In turn, the genes that encode their biosynthesis are also found clustered with the rest of the antibiotic biosynthesis and resistance genes along with the specific glycosyltransferases.

A large percentage of the peptidic natural products that have antibiotic activity are produced by nonribosomal peptide synthetases (NRPSs). The NRPSs catalysts have organizational analogies to the type I polyketide synthases (PKSs) as multidomainal catalysts organized into modules, with multiple modules collected in one or more protein subunits. These include the L-aminoadipyl-L-cysteinyl-D-valine (ACV) tripeptide precursor to the β-lactam families of antibiotics, the channel-forming tyrocidine and gramicidin S, and the topical antibiotic bacitracin. The ACV synthetase has three modules in a polypeptide of 450 kDa, while the heptapeptide scaffold of vancomycin or chloroeremomycin is assembled by three subunits with three, three, and one module, respectively. The enzymatic transformation of the acyclic ACV tripeptide to the bicyclic β-lactam structure of penicillins is catalyzed by a single nonheme FeII enzyme, isopenicillin N synthase (IPNS), that reduces cosubstrate O2 by four electrons to two molecules of water. In the chloroeremomycin cluster there are three hemeproteins, ORFs 7 to 9, and there are two homologs in the bahlimycin cluster that are implicated in genetic knockouts as the cross-linking catalysts. These putative cytochrome P450s could generate the phenolic radicals in the side chains of the heptapeptide substrates to initiate the cross-linkings. In most of the lipopeptide NRPS clusters sequenced to date, the acyltransferase responsible for N-acylation of the N-terminal amino acid does not map with the biosynthetic cluster and little is known about specificity. In the case of mycosubtilin, the first five domains of the MycA subunit are dedicated to construction of the C16-β-NH2-acyl group.

This chapter discusses the enzymatic logic for the formation of certain classes of natural products that have been used in human medicine as antibiotics. The aminoglycoside, or aminocyclitol, antibiotics represent products of secondary carbohydrate metabolism and are prevalent among actinomycetes. Starting with the isolation of streptomycin in 1944, various family members were discovered over the following 25 years, including tobramycin in 1970. Novel aminocyclitols continued to be reported into the 1990s. Two main categories of these carbohydrate antibiotics are exemplified by the streptomycin class and by the 2-deoxystreptamine-containing antibiotics that include neomycins, kanamycins, and gentamicins. The glycoside-to-cyclitol conversion, central to streptomycin antibiotic biosynthetic pathway logic, is found in primary metabolism for the generation of inositol-phosphate from glucose-6- phosphate (glucose-6-P) on the way to phosphoinositide membrane lipid biosynthesis. The prospects for combinatorial biosynthesis to make new aminocyclitols, e.g., with more rings and new connectivities, may be good, setting up the systems for new rounds of semisynthetic alkylations and acylations, although it remains to be seen if useful new activities will result. The bicyclic aminocoumarin ring is constructed from tyrosine, in turn derived from chorismate, the key intermediate in aromatic amino acid biosynthesis. Some of the logic and mechanism of nonribosomal peptide synthetase selection, activation, and modification of amino acid monomers is utilized in these amino acid-based antibiotics.

From a field of research and inquiry that was effectively target poor for the past three decades, there is now at least a temporary embarrassment of target riches, with dozens to hundreds of gene products that are candidates as novel targets. The identification of 150 genes essential for viability in the important pathogen Staphylococcus aureus has been undertaken systematically by expression of antisense ΔRNA to ablate gene function. Some prospects in the traditional validated target areas of cell wall biosynthesis, protein biosynthesis, and DNA replication and repair with certain inhibitors will first have to be noted, followed by pointing out some less traditional targets that command new attention. In addition to the targets that are and will be emerging from the genomics approaches noted at the beginning of this chapter, there are some other enzymes and processes for which there is already reasonable to strong justification for study as novel antibacterial targets. Pyrophosphorylation at the primary alcohol and phosphorylation at the tertiary alcohol by two kinases set up the olefin-forming decarboxylation/P_i elimination reaction to yield the allylic isomer dimethyallyl-pyrophosphate (dimethylallyl-PP) (³). Finally, the isopentenyl-PP isomerase moves the double bond to the (² isomer, isopentenyl-PP, so both isomers are available for elongation reactions.

This chapter talks about synthetic compound libraries and the new approaches with natural products as antibiotic candidates. It summarizes some recent efforts on reprogramming of biosynthetic assembly lines of polyketides (PKs) and nonribosomal peptides (NRPs) assembly lines and of the post-assembly-line enzymatic tailoring reactions to create new variants of natural products, “unnatural natural products." Carrying out combinatorial reprogramming on a large scale requires large numbers of polyketide synthase (PKS) and nonribosomal peptide synthetase (NRPS) gene clusters, an understanding of the rules for cutting and pasting to maximize autonomously folding modules, and rapid methods for gene shuffling. Some of the most intriguing natural products have five-ring sulfur and oxygen heterocycles, thiazoles, and oxazoles, which arise from cyclization of cysteinyl-SH and seryl-OH side chains on the preceding peptide bond carbonyl, catalyzed by cyclization (Cy) domains that are variants of the peptide bond-forming condensation domains. Reconstitution of PKS-NRPS and NRPS-PKS interfaces has been achieved with purified components in vitro to establish the recognition patterns of the KS, C, and Cy catalytic, chain-elongation domains, as a prelude to combinatorial strategies that would make novel hybrid PK-NRP-PK structures.

The emergence of huge population centers, with 10 to 20 million inhabitants in large cities without adequate hygiene and sanitation, has been described as a time bomb for emergence of new infectious diseases. As a counterpoint to newly emerging bacterial pathogens is the increase in known pathogens with new arsenals of drug resistance. While hospitals are clearly fertile arenas for selection of antibiotic-resistant pathogenic bacteria, a complementary arena that has been understood for decades is the use of antibiotics in animal feed, for growth promotion and infectious disease prophylaxis. It has been noted that Clostridium difficile overgrowth has led to restrictions on cephalosporin use in certain geriatric populations and that extensive use of cephalosporins in the 1980s played a significant part in the emergence and spread of Methicillin-resistant S. aureus (MRSA) in London and in Tokyo hospitals as well as the selection for Escherichia coli and Enterobacter cloacae strains with many mutational variants of the plasmid-encoded β-lactamases. Increased molecular knowledge about essential bacterial genes and the ability to screen such validated targets with libraries of new synthetic and natural products are likely to turn up new antibiotics against nontraditional bacterial targets. But new antibiotic molecules by themselves will not alter the kinetics of the cycles of resistance development.