Antimicrobial Chemotherapy, 5th Edition

Part 1 - General Properties of Antimicrobial Agents

Chapter 1

Inhibitors of Bacterial Cell Wall Synthesis

The essence of antimicrobial chemotherapy is selective toxicity—to kill or inhibit the microbe without harming the patient. So far as bacteria are concerned, a prime target for such an attack is the cell wall, since practically all bacteria (with the exception of mycoplasmas) have a cell wall, whereas mammalian cells lack this feature. Several groups of antibiotics, notably β-lactam agents (penicillins, cephalosporins, and their relatives) and glycopeptides (vancomycin and teicoplanin) take advantage of this difference. A few less important antibiotics also act at this level and some compounds used in the treatment of tuberculosis and leprosy act on the specialized mycobacterial cell wall (p. 66).

In general bacterial cell walls conform to two basic patterns, which can be distinguished by that most familiar of all microbiological techniques, the Gram stain. Gram-positive (staphylococci, streptococci, etc.) and Gram-negative (escherichia, pseudomonas, klebsiella, etc.) bacteria respond differently to cell wall active agents and it is helpful to understand the basis for this difference.

Cell wall construction

In both Gram-positive and Gram-negative bacteria the cell wall is formed from a cross-linked chain of alternating units of N-acetylglucosamine and N-acetylmuramic acid, known as peptidoglycan or mucopeptide. The process of synthesis is illustrated in outline inFig. 1.1. N-acetylmuramic acid is manufactured from N-acetylglucosamine by the addition of lactic acid derived from phosphoenolpyruvate. Three amino acids are then added to form a muramic acid tripeptide. Meanwhile, two D-alanine residues, produced from L-alanine by an enzyme called alanine racemase, are joined together by another enzyme, D-alanine synthetase. The linked unit, D-ala-D-ala, is added to the tripeptide and the muramic acid pentapeptide thus formed is joined to an N-acetylglucosamine molecule and passed to a lipid carrier in the cell membrane. The whole building block is transported across the cell membrane and added to the end of the existing cell wall. Finally, adjacent units are cross-linked to give the wall its strength.

 

 

Fig. 1.1 Simplified scheme of bacterial cell wall synthesis showing site of action of cell wall-active antibiotics. (Reproduced fromMedical Microbiology, 16th Edition by David Greenwood (2003), with permission from Elsevier.

 

In Gram-positive organisms the cell wall structure is thick (about 30 nm), tightly cross-linked, and interspersed with polysugarphosphates (teichoic acids), some of which have a lipophilic tail buried in the cell membrane (lipoteichoic acids). Gram-negative bacteria, in contrast, have a relatively thin (2-3 nm), loosely cross-linked peptidoglycan layer and no teichoic acid.

External to the Gram-negative peptidoglycan is a membrane-like structure, composed chiefly of lipopolysaccharide and lipoprotein, which prevents large molecules such as glycopeptides from entering the cell. Small hydrophilic molecules enter Gram-negative bacilli through aqueous channels—called porins—within the outer membrane. Differential activity among some groups of antibiotics, notably the penicillins and cephalosporins, is influenced by their ability to negotiate these porin channels and this, in turn, reflects the size and ionic charge of substituents carried by the individual agents.

β-Lactam antibiotics

Penicillins, cephalosporins, and certain other antibiotics belong to a family of compounds, collectively known as β-lactam antibiotics, which share the structural feature of a β-lactam ring. In the penicillins the β-lactam ring is fused to a five-membered thiazolidine ring, whereas the cephalosporins display a fused β-lactam/dihydrothiazine ring structure (Fig. 1.2). The β-lactam ring is the Achilles' heel of this group of antibiotics because many bacteria possess enzymes (β-lactamases; see p. 130) that are capable of breaking open the ring and rendering the molecule antibacterially inactive.

Penicillins

The original preparations of penicillin were found on analysis to be mixtures of four closely related compounds that were called penicillin F, G, K, and X. Benzylpenicillin (penicillin G), often simply called ‘penicillin’, was chosen for further development because it exhibited the most attractive properties and because a manufacturing process was developed in which Penicillium chrysogenum was persuaded to produce benzylpenicillin almost exclusively.

Early attempts to modify this structure relied on presenting the Penicillium mould used to produce penicillin with different side-chain precursors during the manufacturing process. Later a method was discovered of removing the side-chain of benzylpenicillin to liberate the penicillin nucleus, 6-amino-penicillanic acid (6-APA). Various chemical groupings could then be added to 6-APA according to the ingenuity of the chemist; a large number of compounds, collectively called semisynthetic penicillins, have been prepared in this way.

 

Fig. 1.2 Structures of benzylpenicillin and cephalosporin C, forerunners of the penicillin and cephalosporin groups, respectively. The fused-ring systems and the side-chains, which offer the possibility of modifications introduced in semi-synthetic derivatives, are indicated.

Benzylpenicillin revolutionized the treatment of many potentially lethal bacterial infections, such as scarlet fever, puerperal sepsis, bacterial endocarditis, pneumococcal pneumonia, staphylococcal sepsis, meningococcal meningitis, gonorrhoea, syphilis (and other spirochaetal diseases), anthrax, and many anaerobic infections. The overwhelming importance of benzylpenicillin as a major breakthrough in therapy may be gauged from the fact that it remains today the treatment of choice for all these diseases.

However, resistance has eroded the value of benzylpenicillin. Nearly all staphylococci and many strains of gonococci are now resistant. Moreover, pneumococci exhibiting reduced susceptibility to benzylpenicillin are increasingly prevalent. Such strains are of two types: those for which the minimum inhibitory concentration (MIC) of benzylpenicillin is increased from the usual value of about 0.02 mg/l to 0.1-1 mg/l, and those for which the MIC exceeds 1 mg/l. The former are sufficiently sensitive to enable the antibiotic to be successfully used in high dosage, except in pneumococcal meningitis.

However, penicillin is not clinically reliable in infections with strains exhibiting the higher level of resistance.

Despite its attractive properties benzylpenicillin is not the perfect antimicrobial agent:

  • it exhibits a restricted antibacterial spectrum;
  • it causes hypersensitivity reactions in a small proportion of persons to whom it is given;
  • it is broken down by gastric acidity when administered orally;
  • it is eliminated from the body at a spectacular rate by the kidneys;
  • it is hydrolysed by β-lactamases produced by many bacteria, including staphylococci.

Subsequent developments have been aimed at overcoming these inherent weaknesses while retaining the attractive properties of benzylpenicillin: high intrinsic activity and lack of toxicity.

Acid stability

The first major success in improving the pharmacological properties of penicillin was achieved with phenoxymethylpenicillin (penicillin V). This compound has properties very similar to those of benzylpenicillin, but it is acid stable and thus achieves better and more reliable serum levels when given orally, at the expense of being marginally less active. Azidocillin, phenethicillin, and propicillin exhibit similar properties, but are not widely used.

Prolongation of plasma levels

Most β-lactam antibiotics are rapidly excreted, with plasma half-lives of 1-3 h. Benzylpenicillin is even more rapidly eliminated and several strategies are used in order to maintain effective levels in the body. The blockbuster approach is simply to give enormous doses of this non-toxic drug. Alternatively, oral probenecid can be administered with the penicillin. Probenecid competes for sites of active tubular secretion in the kidney, slowing down the elimination of penicillin. Another solution is to use insoluble derivatives of penicillin. These are injected intramuscularly and act as depots from which penicillin is slowly liberated. Originally, mixtures of penicillin with oily or waxy excipients were used, but insoluble salts, such as procaine penicillin, were later developed. In this way an inhibitory concentration of penicillin can be maintained in the bloodstream for up to 24 h; extremely insoluble salts, such as benzathine penicillin, release penicillin even more slowly, but the concentrations achieved are, of course, correspondingly lower.

 

Extension of spectrum

Broadening the spectrum of benzylpenicillin to encompass Gram-negative bacilli was first achieved by adding an amino group to the side-chain to form ampicillin. Ampicillin is slightly less active than benzylpenicillin against Gram-positive cocci and is equally susceptible to staphylococcal β-lactamase. However, it displays much improved activity against some enterobacteria, including Escherichia coli, Salmonella enterica, and Shigella spp. as well as against Haemophilus influenzae. Oral absorption is relatively poor, but can be improved by esterifying the molecule to form so-called pro-drugs, such as pivampicillin (see p. 198). Such compounds are split by non-specific tissue esterases in the intestinal mucosa to release ampicillin during absorption. Improved absorption has also been more simply achieved by a minor modification to the molecule to produce amoxicillin.

A change of spectrum was brought about by altering the form of the linkage at the 6-position of the penicillanic acid nucleus to amidino (N-CHN) instead of acyl (CO-NH). The only penicillin of this type to become available, mecillinam (known as amdinocillin in the USA), is active against ampicillin-sensitive enterobacteria and some of the more resistant Gram-negative rods. However, mecillinam displays no useful activity against Gram-positive cocci. It is poorly absorbed when given orally, but a pro-drug form, pivmecillinam, can be given by mouth.

Temocillin, a penicillin in which the β-lactam ring carries a methoxy group that renders it stable to most β-lactamases (as in cephamycins; see p. 24), has an unusual spectrum. It is moderately active against many Gram-negative bacilli, but has no useful activity againstPseudomonas aeruginosa, Gram-positive cocci or anaerobic organisms. It was largely abandoned, but a rise in prevalence of Gram-negative bacilli that produce broad-spectrum β-lactamases has prompted its reintroduction.

Antipseudomonal penicillins

None of the agents so far mentioned has any activity against Ps. aeruginosa, an important opportunist pathogen, especially in burns, cystic fibrosis, and immunocompromised patients. A simple carboxyl derivative of benzylpenicillin, carbenicillin, was found to have weak, but useful activity and was used for a time in high dosage. It has been superseded by ticarcillin, the thienyl variant of carbenicillin and by a group of ureido derivatives of ampicillin, including azlocillin and piperacillin. These antipseudomonal penicillins must be administered by injection, but two esterified pro-drugs of carbenicillin, carfecillin, and carindacillin, are available in some countries.

P.19

 

Antistaphylococcal penicillins

By the end of the 1950s, 80% of staphylococci isolated in hospitals were resistant to benzylpenicillin because of their ability to produce penicillinase (β-lactamase). The appearance of these resistant organisms, which often gave rise to serious cross-infection problems, stimulated research into derivatives that were insusceptible to β-lactamase hydrolysis. Success was achieved with methicillin (no longer generally available), nafcillin, and a group called isoxazolylpenicillins: oxacillin, cloxacillin, dicloxacillin, and flucloxacillin. The isoxazolylpenicillins, particularly flucloxacillin, are well absorbed when given orally and are most widely used. They are highly bound to serum protein in the body (see p. 200), but this does not seem to affect their therapeutic efficacy.

Resistance to penicillinase-stable penicillins is caused not by inactivating enzymes, but by alterations to the penicillin target (p. 138). Staphylococci of this type were originally characterized by resistance to methicillin and, although this compound is no longer used in treatment, they are still known as methicillin-resistant staphylococci. Resistance extends to all β-lactam agents and often accompanies resistance to gentamicin and other antibiotics (multiresistant staphylococci). Some strains fully display the resistance phenotype only at a reduced growth temperature or in the presence of high salt concentrations. Particularly troublesome are methicillin-resistant Staphylococcus aureus strains (MRSA), which cause persistent problems in some units; certain strains have a propensity to spread to give rise to miniepidemics (EMRSA).

The spectrum of activity of the most important penicillins in clinical use is shown in Table 1.1.

Penicillins: prescriber's survival kit

  • Benzylpenicillin (penicillin G): original penicillin; still the best against streptococci and spirochaetes
  • Procaine penicillin: depot preparation to prolong plasma levels
  • Phenoxymethylpenicillin: oral variety of benzylpenicillin
  • Amoxicillin (oral)/ampicillin (injection): use if broader spectrum is needed
  • Flucloxacillin: best for staphylococci, except MRSA
  • Piperacillin/ticarcillin: reserve for pseudomonas infections; by injection only

 

 

Table 1.1 Summary of the antibacterial properties of selected penicillins

Penicillin

 

Staphylococci

Streptococci

Neisseriaspp.

Haemophilus influenzae

Enterobacteria

Pseudomonas aeruginosa

Anaerobes

   

Activity

Stabilitya

   

Activity

Stabilitya

 

Benzylpenicillin

 

Very good

Poor

Very good

Very good

Fair

-

-

-

Variable

Phenoxymethylpenicillin

Very good

Poor

Very good

Good

Poor

-

-

-

Variable

Ampicillin

}

Good

Poor

Very good

Very good

Good

Good

Poor

-

Variable

Amoxicillin

Piperacillin

}

Good

Poor

Good

Good

Good

Variable

Variable

Good

Fair

Ticarcillin

Cloxacillin

}

Good

Good

Fair

Fair

Poor

-

-

-

Fair

Flucloxacillin

Mecillinam

 

-

-

-

Fair

Poor

Good

Variable

Poor

Poor

Temocillin

 

-

 

-

Good

Good

Good

Very good

-

-

-, no useful activity.

Stability to β-lactamases of these organisms.

 

 

Cephalosporins

Cephalosporins generally exhibit a somewhat broader spectrum than penicillins, though, idiosyncratically, they lack activity against enterococci. They are mostly stable to staphylococcal β-lactamase and lack crossallergenicity with penicillins (see p. 221).

The original cephalosporin, cephalosporin C, was never marketed, but has given rise to a large family of compounds that continues to expand. The extra carbon atom in the fused ring (Fig. 1.2), offers the possibility of modifications at the carbon designated C-3 (right-hand side of the molecule in Fig. 1.2). Consequently, there are many more cephalosporins than penicillins (Table 1.2). Alterations at either end of the molecule may profoundly affect antibacterial activity but, as a generalization, substituents at the C-3 position have more influence on pharmacokinetic properties. Certain cephalosporins such as cefalotin (no longer widely available) and cefotaxime have an acetoxymethyl side chain at C-3 which is slowly altered by liver enzymes. The altered cephalosporin is usually less active than the parent antibiotic and may display altered pharmacokinetic behaviour, but there is little evidence that the clinical effectiveness is impaired. Several cephalosporins, including cefamandole, cefotetan, cefmenoxime, cefoperazone, and the oxa-cephem latamoxef possess a complex side-chain at the C-3 position that has been implicated in haematological side effects in some patients (see p. 228).

The earliest cephalosporins, cefalotin and cefaloridine, are not absorbed when given orally. Moreover, it soon became clear that the Gram-negative organisms within their spectrum were capable of elaborating a wide variety of enzymes that exhibited potent cephalosporinase activity (see pp. 130-134). As with penicillins, developments within the cephalosporin family were aimed at devising compounds with more attractive properties: oral absorption or other improved pharmacological properties; stability to inactivating enzymes; better intrinsic activity; or a combination of these features.

Cephalosporins are commonly described as first, second, third, or even fourth generation compounds. These loose terms, which are best avoided, refer to:

  • early compounds such as cefalotin and cefalexin that were available before about 1975 (first generation);
  • β-lactamase stable compounds such as cefuroxime and cefoxitin (second generation);
  • compounds such as cefotaxime that combine β-lactamase stability with improved intrinsic activity (third generation);
  • a group of newer compounds that the manufacturers would like to persuade us have special properties (fourth generation).

 

 

Table 1.2 Categorization of cephalosporins in clinical use

Parenteral compounds

 

Oral compounds

   

Cefalotin

Cefacetrile

Ceforanide

Cefalexina

Cefaloglycin

Cefroxadine

Cefaloridine

Cefapirin

Cefonicid

Cefradinea

Cefadroxila

Cefatrizine

Cefazolin

Cefazedone

Ceftezole

Cefaclora

Cefprozila

Loracarbefb

Cefamandole

         

Compounds with improved β-lactamase stability

Compounds with improved β-lactamase stability

Cefuroximea

Cefmetazole

Cefotiam

Non-esterified

Esterified

 

Cefoxitin

Cefotetan

Cefminox

Cefiximea

Cefuroxime axetila

 
     

Ceftibuten

Cefpodoxime proxetila

 

Compounds with improved intrinsic activity and β-lactamase stability

Cefdinir

Cefetamet pivoxil

 
 

Cefteram pivoxil

 

Cefotaximea

Cefmenoxime

Latamoxefc

 

Cefotiam hexetil

 

Ceftriaxonea

Ceftizoxime

Flomoxefc

 

Cefditoren pivoxil

 

Cefodizime

Cefuzonam

   

Cefcapene pivoxil

 

Compounds distinguished by activity against Pseudomonas aeruginosa

   

Broad spectrum

Medium spectrum

Narrow spectrum

     

Ceftazidimea

Cefoperazone

Cefsulodin

     

Cefpiromea

Cefpimizole

       

Cefepime

Cefpiramide

       

Compound available in the UK (2006).

Strictly a carbacephem.

Strictly oxa-cephems.

 

 

In fact, the cephalosporins display such diverse properties that they defy any rigid categorization, but it is helpful to distinguish between those (the majority) that have to be administered parenterally and those that can be given orally. Among injectable compounds, it is useful to consider separately those with improved β-lactamase stability and those notable for their antipseudomonal activity (Table 1.2).

Parenteral compounds susceptible to enterobacterial β-lactamases

Cephalosporins in this group are of limited clinical value and have been largely superseded by other derivatives; all have been abandoned in the UK. Cefazolin has the unusual property of being excreted in fairly high concentration in bile; cefamandole exhibits a modestly expanded spectrum. Others, including cefapirin, ceforanide, and cefonicid, offer no discernible advantage over earlier congeners such as cefalotin.

Parenteral compounds with improved β-lactamase stability

An important advance was achieved with the development of cephalosporins that exhibit almost complete stability to the common β-lactamases of enterobacteria such as Esch. coli and Klebsiella aerogenes. The first of these were cefuroxime and cefoxitin, the latter being one of a group of cephalosporins, collectively called cephamycins, which have a β-lactam ring modified by the addition of a stabilizing methoxy substituent. Other cephamycins available in some countries include cefotetan, cefmetazole, and cefminox. The cephamycins are unusual in displaying useful activity against anaerobes of the Bacteroides fragilis group.

These compounds have been overshadowed by the appearance of cephalosporins that combine almost complete stability to most β-lactamases with much improved intrinsic activity. Cefotaxime was the forerunner of this group of compounds, but several others are available: ceftizoxime and cefmenoxime are similar to cefotaxime; ceftriaxone displays a sufficiently long plasma half-life to warrant once-daily administration; cefodizime is said to possess immunomodulating properties.

Latamoxef (moxalactam), which is strictly an oxa-cephem (see below), also displays activity analogous to that of cefotaxime and its relatives, but differs in possessing useful activity against B. fragilis and related anaerobes. However, latamoxef has lost favour owing to toxicity problems and it is no longer widely available.

 

 

Compounds distinguished by antipseudomonal activity

Ps. aeruginosa is not susceptible to most cephalosporins and, as with penicillins, considerable efforts have been made to find derivatives that include this important opportunist pathogen in their spectrum. Ceftazidime, cefpirome, and cefepime add activity against Ps. aeruginosato broad-spectrum activity comparable with that of cefotaxime and its congeners. These compounds have established a useful role in the management of Ps. aeruginosa infections in seriously ill patients. However, the antistaphylococcal activity is suspect and cefpirome may have some advantage in this respect. Cefepime retains activity against some opportunist Gram-negative bacilli that develop resistance to cefotaxime and its relatives.

Among other antipseudomonal cephalosporins, cefoperazone, cefpimizole, and cefpiramide are not distinguished by any unusual activity against other organisms and cefsulodin is extraordinary in being virtually inactive against bacteria other than Ps. aeruginosa.

Oral cephalosporins

Early development of the cephalosporins yielded cefalexin, a compound of modest activity, particularly in terms of its bactericidal action against Gram-negative bacilli, but which is virtually completely absorbed when given orally. Many other oral derivatives are structurally minor variations on the cefalexin theme. Such compounds include cefradine (the properties of which are indistinguishable from those of cefalexin), cefaclor (which is more active against the important respiratory pathogen H. influenzae), cefadroxil (which exhibits a modestly extended plasma half-life) and cefprozil (which exhibits improved intrinsic activity). Loracarbef is a carbacephem (carbon replacing sulphur in the fused-ring structure), but is otherwise structurally identical to cefaclor. Not surprisingly, its properties closely resemble those of cefaclor.

Cefixime and ceftibuten are structurally unrelated to cefalexin. They display much improved activity against most Gram-negative bacilli, but at the expense of antistaphylococcal (and, in the case of ceftibuten, antipneumococcal) activity, which is very poor. Another compound of this type, cefdinir, appears to lack these defects.

The principle of esterification to produce pro-drugs with improved oral absorption has also been applied to cephalosporins. Two such compounds, cefuroxime axetil and cefpodoxime proxetil, are available in the UK; cefteram pivoxil, cefetamet pivoxil, cefotiam hexetil, cefditoren pivoxil, and cefcapene pivoxil are marketed elsewhere. These esters are fairly well absorbed by the oral route and deliver the parent drug into the bloodstream. Cefpodoxime, cefteram, and cefetamet are more active than the others against most organisms within the spectrum, although cefetamet has poor activity against staphylococci.

A summary of the antimicrobial spectrum of the most important cephalosporins is presented in Table 1.3.

Cephalosporins: prescriber's survival kit

  • Cefuroxime: good broad-spectrum work-horse (cefuroxime axetil for oral use)
  • Cefotaxime/ceftriaxone: more active than cefuroxime; save for serious infections
  • Ceftazidime: serious pseudomonas infections only; injectable
  • Cefalexin/cefradine/cefaclor: oral absorption their chief virtue

Other β-lactam agents

In addition to penicillins and cephalosporins, various other compounds display a β-lactam ring in their structure (Fig. 1.3). The cephamycins, the oxa-cephems latamoxef and flomoxef, and the carbacephem loracarbef—all of which share the general properties of cephalosporins—are examples of such structural variants. Fundamentally different are clavulanic acid, a naturally occurring substance obtained fromStreptomyces clavuligerus, and two penicillanic acid sulphones, sulbactam and tazobactam. These compounds have little useful antibacterial activity, but act as β-lactamase inhibitors. They are used in combination with β-lactamase-labile agents with a view to restoring their activity. Partner compounds reflect the manufacturer's interests: clavulanic acid is combined with amoxicillin (co-amoxiclav) or ticarcillin; sulbactam with ampicillin; and tazobactam with piperacillin.

Structurally novel compounds that exhibit antibacterial activity in their own right include the carbapenems (imipenem, meropenem, panipenem, and ertapenem) and aztreonam, one of a group of compounds, collectively known as monobactams, which have a β-lactam ring but no associated fuse-dring system.

The carbapenems are stable to most bacterial β-lactamases, and exhibit the broadest spectrum of all β-lactam antibiotics, with high activity against nearly all Gram-positive and Gram-negative bacteria other than intracellular bacteria such as chlamydiae. Imipenem is readily hydrolysed by a dehydropeptidase located in the mammalian kidney and is administered together with a dehydropeptidase inhibitor, cilastatin. Aztreonam is also β-lactamase stable, but, in contrast to carbapenems, the activity is restricted to aerobic Gram-negative bacteria.

 

Table 1.3 Summary of the spectrum of antibacterial activity of cephalosporins available in the UK (2006)

Cephalosporin

 

Staphylococci

Streptococcia

Neisseria spp.

Haemophilus influenzae

Enterobacteria

Pseudomonas aeruginosa

Bacteroidesspp.

Cefuroxime

 

Good

Very good

Good

Good

Good

-

-

Cefotaxime

}

Good

Very good

Very good

Very good

Very good

Poor

Poor

Ceftriaxone

Ceftazidime

 

Fair

Good

Very good

Very good

Very good

Good

Poor

Cefpirome

 

Good

Very good

Very good

Very good

Very good

Good

-

Cefalexin

}

             

Cefradine

Good

Good

Poor

Poor

Variable

-

-

Cefadroxil

             

Cefaclor

 

Good

Good

Fair

Good

Variable

-

-

Cefixime

 

Poor

Very good

Good

Good

Very good

-

-

Cefprozil

 

Good

Good

Very good

Very good

Very good

-

-

Cefpodoxime

 

Good

Very good

Very good

Good

Very good

-

-

-, no useful activity.

Enterococci are resistant to all cephalosporins.

 

 

Fig. 1.3 Basic molecular structures of β-lactam antibiotics currently available (examples in parentheses).

Factors affecting β-lactam agents

Penicillins and other β-lactam antibiotics are categorized as bactericidal agents, but this is true only when bacteria are actively dividing. Moreover, the way bacteria respond to β-lactam antibiotics is affected by subtle differences in the mode of action. Several other features of the response that may sometimes have therapeutic implications have also been discovered.

Mode of action of β-lactam agents

All β-lactam antibiotics interfere with the final cross-linking reaction that gives the cell wall its strength (Fig. 1.1). However, several forms of the enzyme that performs this reaction are needed to maintain the complex molecular architecture of the cell and these are differentially inhibited by various β-lactam agents. These target enzymes belong to a group of proteins to which penicillin and other β-lactam antibiotics bind (penicillin-binding proteins; PBPs). Esch. coli, the best-studied species, has seven of these proteins, numbered 1a, 1b, 2, 3, 4, 5, and 6 in order of decreasing molecular weight. PBPs 4-6 are thought to be unconnected with the antibacterial effect of β-lactam agents, since mutants lacking these proteins do not seem to be disabled in any way. Binding to the remainder has been correlated with the various morphological effects of β-lactam antibiotics on Gram-negative bacilli. Thus, cefalexin and its close congeners, as well as aztreonam, bind almost exclusively to PBP 3 and inhibit the division process, causing the bacteria to grow as long filaments. The amidinopenicillin, mecillinam, binds preferentially to PBP 2 and causes a generalized effect on the cell wall so that the bacteria gradually assume a spherical shape. Most other β-lactam antibiotics bind to PBPs 1-3 and, in sufficient concentration, induce the formation of osmotically fragile, wall-deficient forms (called spheroplasts), which typically emerge at the cell wall growth site as the cell starts to divide. The morphological events are illustrated in Fig. 1.4. An important consequence of differences in binding is that compounds such as cefalexin, aztreonam, and mecillinam, which bind only to PBP 3 or PBP 2, are much more slowly bactericidal to Gram-negative bacilli that those that bind PBPs 1, 2, and 3.

In Gram-negative bacilli, rupture of spheroplasts can be quantitatively prevented by raising the osmolality of the growth medium, so cell death appears to be an osmotic phenomenon. The lethal event in Gram-positive organisms, which have much thicker cell walls, appears to be autolysis triggered by the release of lipoteichoic acid following exposure to β-lactam antibiotics.

Optimal dosage effect

A further complication in Gram-positive organisms is that increasing the concentration of β-lactam antibiotics often results in a reduced bactericidal effect. The mechanism of this effect (known as the Eagle phenomenon after its discoverer) is obscure, but may be related to the multiple sites of penicillin action and the fact that cell death occurs only during active growth: saturating a relatively insusceptible penicillin-binding protein may rapidly halt growth and thereby prevent the lethal events that normally follow inhibition of another PBP by lower drug levels.

 

 

Fig. 1.4 Morphological effects of penicillins and cephalosporins on Gram-negative bacilli (scanning electron micrographs): (A) NormalEsch. coli cells; (B) Esch. coli exposed to cefalexin, 32 mg/l, for 1 h; (C) Esch. coli exposed to mecillinam, 10 mg/l, for 2 h; (D) Esch. coli exposed to ampicillin, 64 mg/l, for 1 h, showing lysed debris, central wall lesions, and a spheroplast; higher concentrations of most β-lactam antibiotics cause this effect. (A-C from Greenwood D, O'Grady F, Journal of Infectious Diseases, 1973; 128: 791-4; D from Greenwood D, O'Grady F, Journal of Medical Microbiology, 1969; 2: 435-41.)

Persisters and penicillin tolerance

In both Gram-positive and Gram-negative bacteria, a proportion of the population, called persisters, survive exposure to concentrations of β-lactam antibiotics lethal to the rest of the culture. They remain dormant so long as the antibiotic is present and resume growth when it is removed. In addition, some strains of staphylococci and streptococci display ‘tolerance’ to β-lactam antibiotics in that they succumb much more slowly than usual to the lethal action of β-lactam agents. The therapeutic significance, if any, of persisters is unknown, but penicillin tolerance has been implicated in therapeutic failures in bacterial endocarditis where bactericidal activity is crucial to the success of treatment (p. 318).

Postantibiotic effect

Much has also been made of laboratory observations that the antimicrobial activity of β-lactam agents may persist for an hour or more after the drug is removed. This effect is not confined to β-lactam agents and is more consistently demonstrated with Gram-positive than with Gram-negative organisms. Theoretically, knowledge of postantibiotic effects might influence the design of dosage regimens, but in practice they are too erratic to be used in this way, even if the laboratory observations could be convincingly shown to have clinical relevance, which is presently not the case.

Glycopeptides: For staphs and streps; use mainly for MRSA

The glycopeptides vancomycin and teicoplanin are complex heterocyclic molecules consisting of a multi-peptide backbone to which are attached various substituted sugars. These compounds bind to acyl-D-alanyl-D-alanine in peptidoglycan, thereby preventing the addition of new building blocks to the growing cell wall (Fig. 1.1). Glycopeptides are too bulky to penetrate the external membrane of Gram-negative bacteria, so the spectrum of activity is virtually restricted to Gram-positive organisms. Acquired resistance used to be uncommon, but resistant strains of enterococci are now widely prevalent and staphylococci exhibiting reduced susceptibility are causing concern. Avoparcin, a glycopeptide formerly used in animal husbandry (now banned in the European Union), has been implicated in generating resistance in enterococci, but human use of glycopeptides is equally important. Some Gram-positive genera, including Lactobacillus spp., Pediococcusspp., and Leuconostoc spp. are inherently resistant to glycopeptides, but these organisms are seldom implicated in disease.

Vancomycin

This antibiotic is widely used for the treatment of infections caused by staphylococci that are resistant to methicillin and other β-lactam antibiotics, and for serious infections with Gram-positive organisms in patients who are allergic to penicillin. It is poorly absorbed when given by mouth and must be given by injection. Oral administration is indicated in the treatment of antibiotic-associated diarrhoea caused by toxigenic strains of Clostridium difficile (p. 302-3), but such use is discouraged because of the fear of undermining the value of this compound by promoting the emergence of resistance in Gram-positive cocci.

Early preparations of vancomycin contained impurities that gave the drug a reputation for toxicity. The purified formulations now available are much safer, but renal and ototoxicity still occur, particularly with high dosage. The drug is given by slow intravenous infusion to avoid ‘red man syndrome’ (p. 220).

Teicoplanin

This is a naturally occurring mixture of several closely related compounds with a spectrum of activity similar to that of vancomycin, although some coagulase-negative staphylococci (Staphylococcus epidermidis etc.) are less susceptible to teicoplanin. Some strains of enterococci that are resistant to vancomycin (those with the VanB phenotype; p. 138-140) retain susceptibility to teicoplanin. Unlike vancomycin, teicoplanin can be administered by intramuscular injection; it also has a much longer plasma half-life than vancomycin and a reduced propensity to cause adverse reactions.

Other cell wall active agents

Bacitracin: Topical use only

Bacitracin is a cyclic peptide antibiotic, made up of about 10 amino acids joined in a ring. It was first obtained from a strain of Bacillus subtilis grown from the infected wound of a 7-year-old girl, Margaret Tracy, in whose honour the antibiotic was named.

The spectrum of activity of bacitracin and related cyclic peptides such as gramicidin and tyrocidine is restricted to Gram-positive organisms. They are too toxic for systemic use but are found in topical preparations. Bacitracin also finds a place in microbiology laboratories in the presumptive identification of Streptococcus pyogenes, which is exquisitely susceptible to its action. Bacitracin acts by preventing regeneration of the lipid carrier in the cell membrane, which is left in an unusable phosphorylated form after transporting cell wall subunits (Fig. 1.1).

Cycloserine: Tuberculosis only

Cycloserine has broad-spectrum, but rather feeble antibacterial activity. It is now used only against multiresistant Mycobacterium tuberculosis (p. 349). The drug bears a structural resemblance to the D-isomer of alanine and inhibits alanine racemase. It also blocks the synthetase enzyme that links two D-ala molecules together before they are inserted into the cell wall (Fig. 1.1). Antituberculosis agents that act on special features of the mycobacterial cell wall are discussed in Chapter 3.

Fosfomycin: Uncomplicated cystitis only

Fosfomycin (Fig. 1.5) is a naturally occurring antibiotic originally obtained from a species of Streptomyces isolated in Spain. It is formulated as the sodium salt for parenteral use, but this is unsuitable for oral administration. It is well tolerated, and the ready emergence of bacterial resistance that is observed in vitro does not appear to have been a major problem in treatment. The trometamol (tromethamine) salt, which is highly soluble, well absorbed, and excreted in high concentration in urine, is preferable to the calcium salt for oral therapy. Although fosfomycin is used for assorted purposes in some countries, it is not sufficiently reliable for serious infections. It is best reserved for uncomplicated cystitis, for which the trometamol salt is well suited.

 

Fig. 1.5 Structure of fosfomycin.

Fosfomycin inhibits the pyruvyl transferase enzyme that brings about the condensation of phosphoenolpyruvate and N-acetylglucosamine in the formation of N-acetylmuramic acid (Fig. 1.1). Gram-positive cocci are less susceptible than Gram-negative rods. The precise level of activity is a matter of dispute, since the in-vitro activity can be manipulated by altering the test medium. Glucose-6-phosphate potentiates the activity against many Gram-negative bacilli by inducing the active transport of fosfomycin into the bacterial cell.