33 Attacking the enemy
antimicrobial agents and chemotherapy
The interactions between host, microbial pathogen and antimicrobial agent can be considered as a triangle, and any alteration in one side will inevitably affect the other two sides (Fig. 33.1). In this chapter, two sides of the triangle will be examined in greater detail:
Figure 33.1 The interactions between antimicrobial agents, microorganisms and the human host can be viewed as a triangle. Any effect on one side of the triangle will have effects on the other two sides.
Laboratory aspects of antimicrobial susceptibility tests and assays will also be outlined. The third side of the triangle, the interactions between microorganisms and the human host, has been considered in detail in the preceding chapters. The concluding part of the present chapter will draw together the three sides of the triangle.
The term ‘selective toxicity’ was proposed by the immunochemist Paul Ehrlich (Box 33.1, Fig. 33.2). Selective toxicity is achieved by exploiting differences in the structure and metabolism of microorganisms and host cells; ideally, the antimicrobial agent should act at a target site present in the infecting organism, but absent from host cells. This is more likely to be achievable in microorganisms that are prokaryotes than in those that are eukaryotes, as the former are structurally more distinct from the host cells. (A comparison of the cellular organization of prokaryotic and eukaryotic cells is given in Ch. 1.) At the other end of the spectrum, viruses are difficult to attack because of their obligate intracellular lifestyle. A successful antiviral agent must be able to enter the host cell, but inhibit and damage only a virus-specific target. The desirable features of ideal antimicrobial agents are summarized in Box 33.2.
Box 33.1 Lessons in Microbiology
Just as Pasteur towers over immunomicrobiology, Ehrlich (Fig. 33.2) is the father figure of immunochemistry. His contributions to the science of medicine at all levels are quite extraordinary. He was the first to propose that foreign antigens were recognized by ‘side-chains’ on cells (1890), a brilliant insight that took 70 years to confirm. He also discovered the mast cell, invented the acid-fast stain for the tubercle bacillus, and devised a method to manufacture and commercialize a strong diphtheria antitoxin. He pioneered the development of antibiotics with his work on ‘606’ (or ‘Salvarsan’), a treatment for syphilis, for which he was denounced by the church for interfering with God’s punishment for sin.
While working on the treatment of infections caused by trypanosomes he set forth the concept of ‘selective toxicity’, as illustrated by the following quote: ‘But, gentlemen, it should be made clear that in general this task is much more complicated than that using serum therapy. These chemical agents, in contrast to the antibodies, may be harmful to the body. When such an agent is given to a sick organism, a difference must exist between the toxicity of this agent to the parasite and its toxicity to the host. We must always be aware of the fact that these agents are able to act on other parts of the body as well as on the parasites.’
Like Pasteur, he had a grasp of the continuum from the whole body to the cell and the three-dimensional structure of molecules, and throughout his life, he stressed the importance of molecular interaction as the basis of all biologic function; this is summed up in his famous maxim corpora non agunt nisi fixata or ‘things do not interact unless they make contact’. A Nobel Prize winner in 1908, his name was systematically eliminated from the records by the Nazi regime on account of his Jewish birth, but he was restored to honour by a reconstruction of his laboratory at the Seventh International Congress of Immunology in Berlin in 1989.
a The desired attribute depends on drug usage. Narrow-spectrum drugs cause less disturbance to normal flora and may contribute less to emergence of antibiotic resistance, whereas broad-spectrum compounds are more useful for empiric therapy and treatment of polymicrobial infections. CSF, cerebrospinal fluid.
The term ‘antibiotic’ has traditionally referred to natural metabolic products of fungi, actinomycetes and bacteria that kill or inhibit the growth of microorganisms. Antibiotic production has been particularly associated with soil microorganisms and, in the natural environment, is thought to provide a selective advantage for organisms in their competition for space and nutrients. Although the majority of antibacterial agents in clinical use today are derived from natural products of fermentation, most are then chemically modified (i.e. semi-synthetic) to improve their antibacterial or pharmacologic properties. However, some agents are totally synthetic (e.g. sulphonamides, quinolones). Therefore, the term ‘antibacterial’ or ‘antimicrobial’ agent is often used in preference to ‘antibiotic’. Agents used against fungi, parasites, and viruses can also be included under antimicrobials, but the terms antifungals, antiprotozoans, anthelmintics, and antivirals are more often used.
The discovery of new antimicrobial agents used to be entirely a matter of chance. Pharmaceutical companies undertook massive screening programmes searching for new soil microorganisms that produced antibiotic activity. In the light of our greater understanding of the mechanisms of action of existing antimicrobials, the processes have become rationalized, searching either for new natural products by target-site-directed screening or synthesizing molecules predicted to interact with a microbial target. Genomic approaches to the identification of novel targets have revolutionized this approach. In addition, knowledge of the crystal structure of the key enzymes involved in viral replication such as protease, reverse transcriptase and helicase leads to the design of new drugs. The steps in a rational design programme are summarized in Box 33.3.
The discovery process of new antimicrobial agents has moved away from the random screening of soil microorganisms towards a rational design programme. From discovery to development and marketing can take up to 15 years and cost US$800 million. This list identifies different steps in this programme (average 10 years).
Some antibacterial agents kill bacteria (bactericidal), while others only inhibit their growth (bacteriostatic). Thus, the bactericidal process is irreversible, while bacteriostasis is reversible. Nevertheless, bacteriostatic agents are successful in the treatment of some infections because they prevent the bacterial population from increasing and host defence mechanisms can consequently cope with the static population. However, in immunocompromised patients, bacteriostatic drugs may be less efficacious, and certain infections (e.g., endocarditis) require a bactericidal drug even in an immunocompetent patient.
As a means of classification, the distinction between bactericidal and bacteriostatic agents is blurred because some agents are capable of killing some species, but are only bacteriostatic for others, e.g. chloramphenicol inhibits growth of Escherichia coli, but kills Haemophilus influenzae.
A convenient way of classifying antibacterials is on the basis of their site of action. This classification does not allow an accurate prediction of which antibacterials will be active against which bacterial species, but it does help in the understanding of the molecular basis of antibacterial action, and conversely in the elucidation of many of the synthetic processes in bacterial cells. The five main target sites for antibacterial action are:
These targets differ to a greater or lesser degree from those in the host (human) cells and so allow inhibition of the bacterial cell without concomitant inhibition of the equivalent mammalian cell targets (selective toxicity).
Each target site encompasses a multitude of synthetic reactions (enzymes and substrates), each of which may be specifically inhibited by an antibacterial agent. A range of chemically diverse molecules may inhibit different reactions at the same target site (e.g. protein synthesis inhibitors).
Classification based on chemical structure alone is not of practical use, because there is such diversity. However, a combination of target site and chemical structure provides a useful working classification to organize antibacterial agents into specific families which will be discussed later in this chapter.
Resistance to antibacterial agents is a matter of degree. In the medical setting, we define a resistant organism as one that will not be inhibited or killed by an antibacterial agent at concentrations of the drug achievable in the body after normal dosage. ‘Some men are born great, some achieve greatness, and some have greatness thrust upon them’ (William Shakespeare, Twelfth Night). Likewise, some bacteria are born resistant, others have resistance thrust upon them. In other words, some species are innately resistant to some families of antibiotics because they lack a susceptible target, are impermeable to or enzymatically inactivate the antibacterial agent. The Gram-negative rods with their outer membrane layer exterior to the cell wall peptidoglycan are less permeable to large molecules than Gram-positive cells. However, within species that are innately susceptible, there are also strains that develop or acquire resistance.
In parallel with the rapid development of a wide range of antibacterial agents since the 1940s, bacteria have proved extremely adept at developing resistance to each new agent that comes along. This is illustrated for Staphylococcus aureus by the timeline shown in Figure 33.3. The rapidly increasing incidence of resistance associated with slowing down in the discovery of novel antibacterial agents to combat resistant strains is now recognized worldwide as a serious threat to the treatment of life-threatening infections.
• a single chromosomal mutation in one bacterial cell resulting in the synthesis of an altered protein: for example, streptomycin resistance via alteration in a ribosomal protein, or the single amino acid change in the enzyme dihydropteroate synthetase resulting in a lowered affinity for sulphonamides. A mutational event could also alter (i.e., increase or decrease) the production of a protein resulting in increased resistance.
In the presence of antibiotic, these spontaneous mutants have a selective advantage to survive and outgrow the susceptible population (Fig. 33.4A). They can also spread to other sites in the same patient or by cross-infection to other patients and therefore become disseminated. Chromosomal mutations are relatively rare events (i.e. usually found once in a population of 106–108 organisms) and generally provide resistance to a single class of antimicrobials (i.e. ‘cross-resistance’ to structurally related compounds).
Figure 33.4 A chromosomal mutation (A) can produce a drug-resistant target, which confers resistance on the bacterial cell and allows it to multiply in the presence of antibiotic. Resistance genes carried on plasmids (B) can spread from one cell to another more rapidly than cells themselves divide and spread. Resistance genes on transposable elements (C) move between plasmids and the chromosome and from one plasmid to another, thereby allowing greater stability or greater dissemination of the resistance gene.
Genes on transmissible plasmids may result in resistance to different classes of antimicrobial agents (multiple resistance)
Not content with surviving the antibacterial onslaught by relying on random chromosomal mutation, bacteria are also able to acquire resistance genes on transmissible plasmids (Fig. 33.4B; see also Ch. 2). Such plasmids often code for resistance determinants to several unrelated families of antibacterial agents. Therefore a cell may acquire ‘multiple’ resistance to many different drugs (i.e. in different classes) at once, a process much more efficient than chromosomal mutation. This so-called ‘infectious resistance’ was first described by Japanese workers studying enteric bacteria, but is now recognized to be widespread throughout the bacterial world. Some plasmids are promiscuous, crossing species barriers, and the same resistance gene is therefore found in widely different species. For example, TEM-1, the most common plasmid-mediated beta-lactamase in Gram-negative bacteria, is widespread in E. coli and other enterobacteria and also accounts for penicillin resistance in Neisseria gonorrhoeae and ampicillin resistance in H. influenzae.
Resistance genes may also occur on transposons; the so-called ‘jumping genes’, which by a replicative process are capable of generating copies which may integrate into the chromosome or into plasmids (see Ch. 2). The chromosome provides a more stable location for the genes, but they will be disseminated only as rapidly as the bacteria divide. Transposon copies moving from the chromosome to plasmids are disseminated more rapidly. Transposition can also occur between plasmids, for example, from a non-transmissible to a transmissible plasmid, again accelerating dissemination (Fig. 33.4C).
As discussed previously, antibiotic-resistance genes may individually reside on plasmids, the chromosome, or on transposons found in both locations. However, in some instances multiple resistance genes may come together in a structure known as an integron. As shown in Figure 33.5A, the integron encodes a site-specific recombination enzyme (int gene; integrase), which allows insertion (and also excision) of antibiotic-resistance gene ‘cassettes’ (resistance gene plus additional sequences including an ‘attachment’ region) into the integron attachment site (att). In classic operon fashion, a strong integron promoter controls transcription of the inserted genes. Based on their integration mechanism (integrase, etc.), integrons have been organized into different classes found in both Gram-negative and Gram-positive organisms. Whether acting as independent mobile genetic elements or inserted into transposons, integrons are capable of moving into a variety of DNA molecules, the overall hierarchy of which is depicted in Figure 33.5B. With their ability to capture, organize and rearrange different antibiotic-resistance genes, integrons represent an important mechanism for the spread of multiple antibiotic resistance in clinically important microorganisms.
Staphylococcal genes responsible for resistance to the antibiotic methicillin (discussed below) are found in a specialized cassette arrangement termed staphylococcal chromosomal casette mec (SCCmec). SCCmec inserts into a unique target site on the staphylococcal chromosome. The cassette represents a highly recombinogenic region which may not only rearrange internally but also serve as a target for the insertion of other resistance elements (e.g, transposons and plasmids).
Resistance mechanisms can be broadly classified into three main types. These are summarized below, in Table 33.1 and described in more detail where relevant for each antibiotic in later parts of this chapter. Where bacterial mechanisms of antimicrobial resistance have been elucidated, they appear to involve the synthesis of new or altered proteins. As mentioned above, the genes encoding these proteins may be found on plasmids or the chromosome.
The target may be altered so that it has a lowered affinity for the antibacterial, but still functions adequately for normal metabolism to proceed. Alternatively, an additional target (e.g. enzyme) may be synthesized.
These will be described in the relevant parts on these antibiotics.
The following parts of this chapter deal with groups of antibacterial agents based on their target site and chemical structure. In each case, the discussion attempts to summarize the answers to the questions set out in Table 33.2, reviewing the interactions between antibacterial agent and bacteria and between the antibacterial and the host (i.e. two sides of the triangle in Fig. 33.1).
|What is it?||Chemical structure: natural or synthetic product|
|What does it do?||Target site, mechanism of action|
|Where does it go? (and therefore preferred route of administration)||Absorption, distribution, metabolism and excretion of the drug in the body of the host|
|When is it used?||Spectrum of activity and important clinical uses|
|What are the limitations to its use?||Toxicity to the human host; lack of toxicity, i.e. resistance of the bacteria|
|How much does it cost?||Great variation between agents but cost is a serious limitation on availability of some agents in resource-poor countries|
Peptidoglycan, a vital component of the bacterial cell wall (see Ch. 2), is a compound unique to bacteria and therefore provides an optimum target for selective toxicity. Synthesis of peptidoglycan precursors starts in the cytoplasm; wall subunits are then transported across the cytoplasmic membrane and finally inserted into the growing peptidoglycan molecule. Several different stages are therefore potential targets for inhibition (Fig. 33.6). The antibacterials that inhibit cell wall synthesis are varied in chemical structure. The most important of these agents are the beta-lactams, the largest group, and the glycopeptides which are active only against Gram-positive organisms. Bacitracin (primarily used topically) and cycloserine (mainly used as a ‘second-line’ medication for treatment of tuberculosis, discussed later in this chapter) have many fewer clinical applications.
Figure 33.6 The synthesis of peptidoglycan is a complex process that begins in the cytoplasm, proceeds across the cytoplasmic membrane and leads to the attachment of new wall units to the growing peptidoglycan chain. This synthetic pathway can be inhibited at a variety of points by antibacterial agents. The precise mechanism of inhibition caused by glycopeptides such as vancomycin is unknown, but the mechanism of action of beta-lactams has now been fully elucidated (see text). NAG, N-acetyl glucosamine; NAM, N-acetyl muramic acid; UDP, uridine diphosphate.
Beta-lactams contain a beta-lactam ring and inhibit cell wall synthesis by binding to penicillin-binding proteins (PBPs)
Beta-lactams comprise a very large family of different groups of bactericidal compounds, all containing the beta-lactam ring. The different groups within the family are distinguished by the structure of the ring attached to the beta-lactam ring – in penicillins this is a five-membered ring, in cephalosporins a six-membered ring – and by the side chains attached to these rings (Fig. 33.7).
Figure 33.7 The beta-lactam family. The ring structure is common to all beta-lactams and must be intact for antibacterial action. Enzymes (beta-lactamases) that catalyse the hydrolysis of the beta-lactam bond render the agents inactive. The penicillins and cephalosporins are the major classes of beta-lactam antibiotics, but other members of the family, particularly the carbapenems and monobactams, are the focus of new developments.
PBPs are membrane proteins (e.g. carboxypeptidases, transglycosylases and transpeptidases) capable of binding to penicillin (hence the name PBP) and are responsible for the final stages of cross-linking of the bacterial cell wall structure. Inhibition of one or more of these essential enzymes results in an accumulation of precursor cell wall units, leading to activation of the cell’s autolytic system and cell lysis (Fig. 33.8).
Figure 33.8 Penicillin-binding proteins (PBPs) play a key role in the final stages of peptidoglycan synthesis. They catalyse the cross-linkage of wall subunits, which are then incorporated into the cell wall. Beta-lactams are able to enter the cell (e.g. through pores in the outer membrane of Gram-negatives) and bind to the PBP. This prevents it from catalysing the cross-linkage of subunits, leading to their accumulation in the cell and the release of autolytic enzymes, which causes cell lysis. Within the periplasmic space of Gram-negatives (b1) beta-lactamases can inactivate beta-lactams before they reach their target PBPs, thereby protecting the cell from antibiotic action. Alternatively, mutant PBPs fail to bind beta-lactase, thus allowing peptidoglycan synthesis to occur. In Gram-positive bacteria (b2) beta-lactams may be extracellularly destroyed by beta-lactamases or rendered ineffective, as in Gram-negatives, by mutant PBPs.
Although the majority of beta-lactams have to be administered intramuscularly or intravenously, there are some orally active agents. Most achieve clinically useful concentrations in the cerebrospinal fluid (CSF) when the meninges are inflamed (as in meningitis) and the blood–brain barrier becomes more permeable. In general, they are not effective against intracellular organisms.
A few of the cephalosporins, notably cefotaxime, are metabolized to compounds with less microbiologic activity. All beta-lactams are excreted in the urine, and for some, such as benzylpenicillin, this is very rapid; hence the need for frequent doses. Probenecid can be administered concurrently to slow down excretion and maintain higher blood and tissue concentrations for a longer period of time.
Different beta-lactams have different clinical uses, but are not active against species that lack a cell wall
A vast array of beta-lactam antibiotics are currently registered for clinical use. Some, such as penicillin, are active mainly against Gram-positive organisms, whereas others (e.g. semi-synthetic penicillins, carboxypenems, monobactams, second-, third- and fourth-generation cephalosporins) have been developed for their activity against Gram-negative rods. Only the more recent beta-lactams are active against innately more resistant organisms such as Pseudomonas aeruginosa (Table 33.3).
|Drug class||Category||General spectrum of activity|
|Penicillin G, Va||Natural penicillin||Gram-positive bacteria|
|Semisynthetic (beta-lactamase resistant) penicillin||Gram-positive bacteria (incl. beta-lactamase producers)|
|Semisynthetic (amino) penicillin||Gram-positive bacteria|
Gram-negative bacteria, including spirochetes, Listeria monocytogenes, Proteus mirabilis and some Escherichia coli
|Semisynthetic (carboxy) penicillin|
Semisynthetic (ureido) penicillin
Enhanced coverage of Gram-negatives, including Pseudomonas and Klebsiella
|First generation||Gram-positive bacteria|
|Fourth generation||Improved activity against Gram-negative bacteria|
|Ceftaroline||Anti-MRSA||Improved activity, especially against MRSA|
Improved activity against Bacillus fragilis
|Gram-positive and Gram-negative bacteria|
|Aztreonam||Gram-negative bacteria including Haemophilus influenza and Pseudomonas aeruginosa|
Although there are many beta-lactam agents available, the most commonly used ones are listed, together with their main indications.
It is important to remember that beta-lactams are not active against species that lack a cell wall (e.g. Mycoplasma) or those with very impenetrable walls such as mycobacteria, or intracellular pathogens such as Brucella, Legionella and Chlamydia.
Methicillin-resistant staphylococci (e.g. Staph. aureus, Staph. epidermidis – MRSA, MRSE, respectively) synthesize an additional PBP (PBP2a) which has a much lower affinity for beta-lactams than the normal PBPs and is therefore able to continue cell wall synthesis when the other PBPs are inhibited. Although the mecA gene which codes for PBP2a is present on the chromosome in all cells of a resistant population, in many instances it may only be transcribed in a proportion of the cells, resulting in a phenomenon known as ‘heterogeneous resistance’. In the laboratory, special cultural conditions are used to enhance expression and demonstrate resistance. Methicillin-resistant staphylococci commonly produce beta-lactamase (see below) and are resistant to all other beta-lactams with the exception of ceftaroline, the first cephalosporin approved by the US FDA for activity against MRSA. This cephalosporin binds to PBP2a with an affinity 2000-fold better than other beta-lactams, and is thus effective in treating infections caused by MRSA.
Other organisms such as Streptococcus pneumoniae, Neisseria gonorrhoeae and Haemophilus influenzae may also utilize PBP changes to achieve beta-lactam resistance, which may vary depending on the compound employed.
This mechanism is found in Gram-negative cells where beta-lactams gain access to their target PBPs by diffusion through protein channels (porins) in the outer membrane. Mutations in porin genes result in a decrease in permeability of the outer membrane and hence resistance. Strains resistant by this mechanism may exhibit cross-resistance to unrelated antibiotics that use the same porins.
Beta-lactamases are enzymes that catalyse the hydrolysis of the beta-lactam ring to yield microbiologically inactive products. Genes encoding these enzymes are widespread in the bacterial kingdom and are found on the chromosome and on plasmids.
The beta-lactamases of Gram-positive bacteria are released into the extracellular environment (Fig. 33.8A) and resistance will only be manifest when a large population of cells is present. The beta-lactamases of Gram-negative cells, however, remain within the periplasm (Fig. 33.8B).
To date, hundreds of different beta-lactamase enzymes have been described. All have the same function but with differing amino acid sequences that influence their affinity for different beta-lactam substrates. Some enzymes specifically target penicillins or cephalosporins, while others are especially troublesome in broadly attacking most beta-lactam compounds (i.e. extended-spectrum beta-lactamases, ESBLs). Some beta-lactam antibiotics (e.g. carbapenems) are hydrolysed by very few enzymes (beta-lactamase stable), whereas others (e.g. ampicillin) are much more labile. Beta-lactamase inhibitors such as clavulanic acid (Fig. 33.9) are molecules that contain a beta-lactam ring and act as ‘suicide inhibitors’, binding to beta-lactamases and preventing them from destroying beta-lactams. They have little bactericidal activity of their own.
Figure 33.9 Clavulanic acid, a product of Streptomyces clavuligerus, inhibits the most common beta-lactamases (e.g. TEM enzymes) and allows amoxicillin to inhibit cells producing these enzymes. Augmentin is the most widely used of these combination drugs. Other combinations include ticarcillin and clavulanic acid, and piperacillin and tazobactam.
Statistics regarding allergy to beta-lactam drugs are complicated by the fact that the problem historically involves self-reporting by patients who are often mistaken in their ‘diagnosis’. Nevertheless, serious allergy to beta-lactam drugs in the form of an immediate (type 1) hypersensitivity reaction may occur in ca. 0.5–4% of patients, although anaphylaxis occurs much less frequently (ca. 0.004 to 0.04% of penicillin treatment courses). Mild idiopathic reactions, usually in the form of a rash, are more common (ca. 25% of treatment courses), especially with ampicillin. Patients who are allergic to penicillin are often allergic to cephalosporins (less with third-generation compounds) and vice versa, but aztreonam, a monobactam, shows negligible cross-reactivity.
Neurotoxicity and seizures can occur with all the beta-lactams if improperly dosed for body weight and kidney function, especially in patients with renal impairment. This toxicity is manifest as fits, unconsciousness, myoclonic spasms and hallucinations. Carbenicillin can cause platelet dysfunction and sodium overload (because it is given as a sodium salt), especially in patients with liver failure, renal failure and congestive heart failure.
Glycopeptides include vancomycin and teicoplanin. Both are very large molecules and therefore have difficulty penetrating into Gram-negative cells. Teicoplanin is a natural complex of five different but closely related molecules.
Glycopeptides are bactericidal and interfere with cell wall synthesis by binding to terminal d-alanine-d-alanine at the end of pentapeptide chains that are part of the growing bacterial cell wall structure (see Fig. 33.6). This binding inhibits the transglycosylation reaction and prevents incorporation of new subunits into the growing cell wall. As glycopeptides act at an earlier stage than beta-lactams, it is not useful to combine glycopeptides and beta-lactams in the treatment of infections.
Vancomycin and teicoplanin are not absorbed from the gastrointestinal tract and do not penetrate the CSF in patients without meningitis. However, bactericidal concentrations are achieved in most patients with meningitis because of the increased permeability of the blood–brain barrier. Excretion is via the kidney.
• the treatment of infections caused by Gram-positive cocci and Gram-positive rods that are resistant to beta-lactam drugs, particularly multiresistant Staphylococcus aureus and Staphylococcus epidermidis
• the treatment of Clostridium difficile in antibiotic-associated colitis, although concerns that this may promote emergence of glycopeptide-resistant enterococci in the gut flora have led to the increasing use of alternative compounds.
As mentioned previously, Gram-negative bacteria are ‘naturally’ resistant to the glycopeptides, since these compounds are too large to efficiently move through the outer membrane to the peptidoglycan. Other organisms have an altered glycopeptide target, such as pentapeptides, terminating in d-alanine-d-lactate (e.g. Erysiplothrix, Leuconostoc, Lactobacillus and Pediococcus) or d-alanine-d-serine (e.g. Enterococcus gallinarum, Enterococcus casseliflavus).
Historically, the most clinically relevant acquired glycopeptide resistance has been observed in Enterococcus faecium and Enterococcus faecalis (vancomycin-resistant enterococci; VRE), first reported by investigators in the UK in 1986. Since that time, a variety of resistance phenotypes have been described which can be differentiated by transferability (e.g. plasmid association), inducibility and extent of resistance (Table 33.4). The genes associated with the highest levels of glycopeptide resistance are vanA, vanB, and vanD which encode a ligase producing pentapeptides terminating in d-alanine-d-lactate.
VanA-type glycopeptide resistance has been the most extensively studied and is characterized by inducible high-level resistance to both vancomycin and teicoplanin. VanA is associated with transposable elements related to Tn1546 (ca. 11 kb in size) which may be chromosomal or plasmid (transferable) in nature.
VanB is associated with inducible high-level resistance to vancomycin but not teicoplanin (although teicoplanin resistance can be induced by prior exposure to vancomycin). VanB resistance may be chromosomal or plasmid linked and is associated with very large transposable elements such as Tn1549 (34 kb).
Glycopeptide resistance in the staphylococci occurs by mutation or by acquisition from the enterococci
Within the coagulase-negative staphylococci (CNS), Staphylococcus epidermidis and Staphylococcus haemolyticus are especially prone to development of glycopeptide resistance by mechanisms which remain incompletely understood. Nevertheless, resistant clinical and laboratory-generated isolates have been shown to differ from their susceptible counterparts in a variety of ways including changes in glycopeptide binding capacity, membrane proteins and cell wall synthesis and composition.
Coagulase-positive staphylococci (i.e. Staphylococcus aureus) showing decreased susceptibility to glycopeptides (but not fully resistant) were first described by Japanese investigators in 1996. The reduced susceptibility of these vancomycin-intermediate or glycopeptide-intermediate isolates (VISA or GISA, respectively) may be either homogeneously or heterogeneously expressed. In either case, ‘resistance’ is not associated with VanA, B, or D but, instead, appears to involve other mechanisms affecting cell wall composition (e.g. leading to increased thickness, etc.).
Vancomycin is usually given by intravenous infusion, administered slowly to avoid ‘red-man’ syndrome due to histamine release. Particular care must be taken to prevent toxic concentrations accumulating in patients with renal impairment. Oral vancomycin is used for treatment of antibiotic-associated pseudomembranous colitis due to Clostridium difficile. Teicoplanin is less toxic than vancomycin and can be given by intravenous bolus and by intramuscular injection.
Although protein synthesis proceeds in an essentially similar manner in prokaryotic and eukaryotic cells, it is possible to exploit the differences (e.g. 70 S vs 80 S ribosome) to achieve selective toxicity. The process of translation of the messenger RNA (mRNA) chain into its corresponding peptide chain is complex, and a range of antibacterial agents act as inhibitors, although the full details of their mechanisms of action are not yet known (Fig. 33.10).
Figure 33.10 The synthetic pathway leading to the production of new protein in bacterial cells is extremely complex and still not fully elucidated. A number of different groups of antibacterial agents act by inhibiting proteins with specific reactions in this synthetic pathway. They can be grouped into those that act on the 30 S subunit of the ribosome (e.g. aminoglycosides and tetracyclines) and those that act on the 50 S subunit (e.g. chloramphenicol, lincosamides, erythromycin and fusidic acid). fmet-tRNA, formylmethionyl-transfer RNA.
The aminoglycosides contain either streptidine (streptomycin) or 2-deoxystreptamine (e.g. gentamicin; Table 33.5). The original structures have been modified chemically by changing the side chains to produce molecules such as amikacin and netilmicin that are active against organisms that have developed resistance to earlier aminoglycosides.
|Gentamicina||Complex of 3 closely related structures; first aminoglycoside with broad spectrum|
|Tobramycinb||Activity very similar to gentamicin but slightly better against Pseudomonas aeruginosa|
|Amikacin||Semi-synthetic derivative of kanamycin; active against many gentamicin- resistant Gram-negative rods|
|Netilmicinb||Activity spectrum similar to amikacin: possibly lower toxicity|
|Neomycinb||Too toxic for parenteral use but has topical uses in decontaminating mucosal surfaces|
|Streptomycinb||Oldest aminoglycoside; now use restricted to treatment of tuberculosis|
They are also differentiated by the genus of microorganisms that produces them, and this is reflected in the spelling of the names.
Aminoglycosides act by binding to specific proteins in the 30 S ribosomal subunit, where they interfere with the binding of formylmethionyl-transfer RNA (fmet-tRNA) to the ribosome (Fig. 33.10), thereby preventing the formation of initiation complexes from which protein synthesis proceeds. In addition, aminoglycosides cause misreading of mRNA codons and tend to break apart functional polysomes (protein synthesis by multiple ribosomes tandemly attached to a single mRNA molecule) into non-functional monosomes.
Aminoglycosides are not absorbed from the gut, do not penetrate well into tissues and bone, and do not cross the blood–brain barrier. Thus, they are usually administered as an intravenous infusion. Intrathecal administration of streptomycin is used in the treatment of tuberculous meningitis, and gentamicin may be administered by this route in the treatment of Gram-negative meningitis in neonates. Aminoglycosides are excreted via the kidney.
Gentamicin, tobramycin, amikacin and netilmicin are important for the treatment of serious Gram-negative infections, including those caused by P. aeruginosa (Box 33.4). They are not active against streptococci or anaerobes, but are active against staphylococci. Against P. aeruginosa, amikacin is most active. Amikacin and netilmicin may be active against strains resistant to gentamicin and tobramycin (see below). Streptomycin is now reserved almost entirely for the treatment of mycobacterial infections. Neomycin is not used for systemic treatment, but can be used orally in gut decontamination regimens in neutropenic patients.
Aminoglycosides are valuable additions to the clinician’s armamentarium despite their potential toxicity. They are important agents active against Gram-negative facultative bacteria and are often used in combination with beta-lactams to broaden the spectrum to include streptococci and some anaerobes, which are not susceptible to aminoglycosides alone. Resistance to aminoglycosides, particularly among enterobacteria and staphylococci, is mediated by the production of aminoglycoside-modifying enzymes, which react with groups on the aminoglycoside molecule to yield an altered aminoglycoside product. This competes with the unmodified aminoglycoside for uptake into the cell and binding to the ribosome.
Production of aminoglycoside-modifying enzymes is the principal cause of resistance to aminoglycosides
Although relatively uncommon, resistance to aminoglycoside antibiotics may occur by alteration of the 30 S ribosomal target protein (e.g. a single amino acid change in the P12 protein prevents streptomycin binding). Resistance may also arise through alterations in cell wall permeability or in the energy-dependent transport across the cytoplasmic membrane.
Production of aminoglycoside-modifying enzymes is the most important mechanism of acquired resistance (Fig. 33.11). The genes for these enzymes are often plasmid-mediated, located on transposons, and transferable from one bacterial species to another. The enzymes alter the structure of the aminoglycoside molecule, thus inactivating the drug. The type of enzyme determines the spectrum of resistance of the organism containing it.
Figure 33.11 Prototype structure of aminoglycoside consisting of aminohexoses linked via glycosidic linkage to a central 2-deoxystreptamine nucleus. Hydroxyl and amino groups are sites at which these compounds can be inactivated by phosphorylation, adenylation or acetylation catalysed by enzymes produced by resistant strains.
The therapeutic ‘window’ between the serum concentration of aminoglycoside required for successful treatment and that which is toxic is small. Blood concentrations should be monitored regularly, particularly in patients with renal impairment. Netilmicin is reported to be one of the less toxic aminoglycoside antibiotics.
Tetracyclines are bacteriostatic compounds that differ mainly in their pharmacological properties rather than in their antibacterial spectra
Figure 33.12 Tetracyclines are four-ring molecules with five different sites for substitution, thereby giving rise to a family of molecules with different substituents at different sites. Members of the family differ more in their pharmacologic properties than in their spectrum of activity.
Tetracyclines inhibit protein synthesis by binding to the small ribosomal subunit in a manner that prevents aminoacyl transfer RNA from entering the acceptor sites on the ribosome (see Fig. 33.10). While this process may occur with both prokaryotic and eukaryotic ribosomes, the selective action of tetracyclines is due to their much greater uptake by prokaryotic cells.
Tetracyclines are usually administered orally. Doxycycline and minocycline are more completely absorbed than tetracycline, oxytetracycline and chlortetracycline and so result in higher serum concentrations and less gastrointestinal upset because there is less inhibition of normal gut flora. Tetracyclines are well distributed and penetrate host cells to inhibit intracellular bacteria. They are excreted primarily in bile and urine.
Tetracyclines are active against a wide variety of bacteria, but their use is restricted due to widespread resistance
Tetracyclines are used in the treatment of infections caused by mycoplasmas, chlamydiae and rickettsiae. Resistance in other genera is common, due partly to the widespread use of these drugs in humans and also to their use as growth promoters in animal feed. The resistance genes are carried on a transposon, and new cytoplasmic membrane proteins are synthesized in the presence of tetracycline. As a result, tetracycline is positively pumped out of resistant cells (efflux mechanism). Although included with the tetracyclines (Fig. 33.12), tigecycline is a new member of a related class of compounds (glycylcyclines), derived from minocycline, with activity against bacteria resistant to tetracyclines.
Tetracyclines suppress normal gut flora, resulting in gastrointestinal upset and diarrhea and encouraging overgrowth by resistant and undesirable bacteria (e.g. Staph. aureus) and fungi (e.g. Candida).
Interference with bone development and brown staining of teeth occurs in the fetus and in children. Systemic administration may cause liver damage. The potential for photosensitization is another caveat associated with the use of tetracyclines in all patients.
Chloramphenicol contains a nitrobenzene nucleus and prevents peptide bond synthesis, with a bacteriostatic result
Chloramphenicol is a relatively simple molecule containing a nitrobenzene nucleus, which is responsible for some of the toxic problems associated with the drug (see below). Other derivatives have been produced, but none is in widespread clinical use.
Chloramphenicol has affinity for the large (50 S) ribosomal subunit where it blocks the action of peptidyl transferase, thereby preventing peptide bond synthesis (see Fig. 33.10). The drug has some inhibitory activity on human mitochondrial ribosomes (which are also 70 S) which may account for some of the dose-dependent toxicity to bone marrow (see below).
Chloramphenicol is well absorbed when given orally, but can be given intravenously if the patient cannot take drugs by mouth. Topical preparations are also available. It is well distributed in the body and penetrates host cells. Chloramphenicol is metabolized in the liver by conjugation with glucuronic acid to yield a microbiologically inactive form that is excreted by the kidneys.