Attacking the enemy: antimicrobial agents and chemotherapy

33 Attacking the enemy


antimicrobial agents and chemotherapy




Selective toxicity


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 imageLessons in Microbiology



Paul Ehrlich (1854–1915)


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.





Discovery and design of antimicrobial agents


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.




Classification of antibacterial agents


There are three ways of classifying antibacterial agents:








Resistance to antibacterial agents


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.



The genetics of 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.




Chromosomal mutation may result in resistance to a class of antimicrobial agents (cross-resistance)


Resistance may arise from:



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).




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.




‘Cassettes’ of resistance genes may be organized into genetic elements called integrons


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.







Inhibitors of cell wall synthesis


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.




Beta-lactams



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).



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).





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).


Table 33.3 Characteristics of representative beta-lactams











































































Drug class Category General spectrum of activity
Penicillins    
Penicillin G, Va Natural penicillin Gram-positive bacteria
Cloxacillina
Dicloxacillina
Nafcillina
Oxacillina
imageSemisynthetic (beta-lactamase resistant) penicillin imageGram-positive bacteria (incl. beta-lactamase producers)
Amoxicillina,b
Ampicillina,b
imageSemisynthetic (amino) penicillin Gram-positive bacteria
Gram-negative bacteria, including spirochetes, Listeria monocytogenes, Proteus mirabilis and some Escherichia coli
Carbenicillina
Ticarcillinb
Mezlocillin
Piperacillinb
Semisynthetic (carboxy) penicillin
Semisynthetic (ureido) penicillin
Gram-positive bacteria
Enhanced coverage of Gram-negatives, including Pseudomonas and Klebsiella
Cephalosporins    
Cefadroxila
Cefazolin
Cephalexina
Cephalothin
Cephradinea
imageFirst generation imageGram-positive bacteria
Cefaclora
Cefamandole
Cefonicid
Cefprozila
Cefuroximea
imageSecond generation  
Cefdinira
Cefditorena
Cefoperazone
Cefpodoximea
Cefotaxime
Ceftazidime
Ceftibutena
Ceftizoxime
Ceftriaxone
imageThird generation  
Cefepime
Cefpirome
imageFourth generation Improved activity against Gram-negative bacteria
Ceftaroline Anti-MRSA Improved activity, especially against MRSA
Cephamycinc    
Cefmetazole
Cefotetan
Cefoxitin
  Gram-positive bacteria
Improved activity against Bacillus fragilis
Carbapenems    
Ertapenem
Imipenem
Meropenem
Doripenem
  Gram-positive and Gram-negative bacteria
Monobactams    
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.


a Oral formulation available.


b Can be formulated in combination with beta-lactamase inhibitors (see Fig. 33.9).


c Often classified with second generation cephalospoxins.


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.



Resistance to beta-lactams may involve one or more of the three possible mechanisms





Resistance by production of beta-lactamases


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.





Glycopeptides






Resistance








Inhibitors of protein synthesis


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).




Aminoglycosides



The aminoglycosides are a family of related molecules with bactericidal activity


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.


Table 33.5 Aminoglycoside-aminocyclitol antibiotics classified according to their chemical structure



























4,6-distributed 2-deoxystreptamines
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
4,5-disubstituted 2-deoxystreptamines
Neomycinb Too toxic for parenteral use but has topical uses in decontaminating mucosal surfaces
Streptidine-containing
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.


a Micins from Micromonospora species.


b Mycins from Streptomyces species.


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.




Gentamicin and the newer aminoglycosides are used to treat serious Gram-negative infections


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.






Tetracyclines






Chloramphenicol


Jul 9, 2017 | Posted by in MICROBIOLOGY | Comments Off on Attacking the enemy: antimicrobial agents and chemotherapy
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