Antimicrobial Chemotherapy, 4th Edition

Resistance to antimicrobial agents

12

Mechanisms of acquired resistance

  1. J. Towner

Three conditions must be met in order that a particular antimicrobial agent can inhibit sensitive bacteria:

  • a vital target susceptible to the action of a low concentration of the antibiotic must exist in the bacterial cell
  • the antibiotic must be able to reach the target
  • the antibiotic must not be inactivated before binding to the target.

The targets of individual antibiotics are often enzymes or other essential proteins. Most antimicrobial agents have to pass through the cell wall and outer membranes to reach their target, and many are carried into the cell by active transport mechanisms that are occupied more normally in transporting sugars and other beneficial substances.

Some of the differences in susceptibility of bacterial species are related to differences in cell wall structure. For example, the cell envelope of Gram-negative bacteria is a more complex structure than the Gram-positive cell wall and offers a relatively greater barrier to many antibiotics, including penicillins and macrolides. Polymyxins exert their effects at the cell surface by disrupting the Gram-negative cell membranes from the outside in a way which resembles the action of some detergents.

The mechanisms by which resistance can arise can be divided into four major groups:

  • destruction or inactivation of the antibiotic
  • alteration of the target site to reduce or eliminate binding of the antibiotic to the target
  • reduction in cell surface permeability or blockage of the mechanism by which the antibiotic enters the cell
  • acquisition of a replacement for the metabolic step inhibited by the antibiotic.

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It is worth emphasising that certain resistance mechanisms overlap within these groups. Some of the known mechanisms of antibiotic resistance are summarized in Table 12.1.

Table 12.1 Important known resistance mechanisms for the major groups of antibiotics

 

Inactivation or modification

Altered target site

Reduced permeability or access

Metabolic by-pass

 

β-Lactam antibiotics
Chloramphenicol
Aminoglycosidesa

β-Lactam antibiotics
Streptomycin
Chloramphenicol
Erythromycinb
Fusidic acid
Quinolones
Rifampicin
Glycopeptidesb

Tetracyclinesc
β-Lactam antibiotics
Chloramphenicol
Quinolonesc
Aminoglycosides

Trimethoprim
Sulphonamides

 

aResulting in reduced drug uptake.

bResulting from enzymic modification.

cResulting from an increased efflux.

Inactivation or modification mechanisms

These are probably the most important resistance mechanisms to be met in clinical practice since they include the common modes of resistance to penicillins and cephalosporins, the therapeutic agents of widest use.

β-Lactam antibiotics

There are many different agents in this group (see Chapter 1) and a correspondingly large number of β-lactamases which catalyse hydrolysis of the β-lactam ring to form an inactive product (Fig. 12.1). In addition, varying levels of β-lactamase production, as well as differences in permeability of the Gram-negative cell envelope, play a considerable part in determining the differential susceptibilities of bacteria to this group of antibiotics.

 

Fig. 12.1 β-Lactamase hydrolysis of penicillin to form the corresponding penicilloic acid, which is antibacterially inactive. Cephalosporins may be attacked in a similar fashion, but the resultant cephalosporoic acid is usually unstable and disintegrates into smaller fragments.

All bacteria appear to contain enzymes capable of hydrolysing β-lactam antibiotics. Indeed, it has been suggested that the normal function and evolutionary origin of β-lactamases is to break a β-lactam structure that is a transitory intermediate in cell wall synthesis. These inherent enzymes are encoded by the chromosome, and are normally bound closely to the cell membrane. In general, they are produced only in small amounts, they attack cephalosporins more readily than penicillins, and they act relatively slowly. In certain organisms, notably

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Enterobacter spp., Acinetobacter spp., Citrobacter spp., and Pseudomonas aerug-inosa, gross overproduction of these chromosomal enzymes has been associated with treatment failures, even with ostensibly ‘β-lactamase-stable’ cephalosporins and carbapenems. Resistance seems to result from the slow hydrolysis mediated by these enzymes, combined with cell wall mutations that partially impede entry of the antibiotics into the bacterial cell.

From a clinical point of view, most interest centres on the large number of plasmid-encoded enzymes which are the major cause of bacterial resistance to penicillins and cephalosporins in clinical isolates. Some are located on trans-posons (described more fully in Chapter 13), which allow movement of genes between plasmids and the chromosome, so that the distinction between plasmid-encoded and chromosomally-encoded enzymes can sometimes become blurred. The plasmid-encoded β-lactamases of Gram-negative bacilli embrace a wide variety of types which are physico-chemically quite distinct.

Among Gram-positive cocci, plasmid-encoded β-lactamases of clinical significance are found almost exclusively in staphylococci. These enzymes rapidly hydrolyse benzylpenicillin, ampicillin, and most other penicillins, but are less active against ‘antistaphylococcal penicillins’ (p. 19) and cephalosporins. Staphylococcal β-lactamases are inducible exoenzymes conforming to a few biochemical types which are probably related closely. In streptococci, β-lactamases are usually absent, and these bacteria have consequently remained, with few exceptions, susceptible to benzylpenicillin.

Various characters are used to distinguish the different enzymes. These include substrate profile, and the action of enzyme inhibitors such as clavulanic acid (p. 24) and the ion-chelator, ethylenediaminetetraacetic acid (EDTA). Substrate profile refers to the hydrolytic activity of a β-lactamase preparation against a number of β-lactam substrates, often expressed as the ratio to a value for a reference substrate such as benzylpenicillin.

By these and newer techniques based on use of DNA : DNA hybridization or the polymerase chain reaction (PCR) to recognize specific genes, it has been

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possible to distinguish a large number of different plasmid-encoded β-lactamases in Gram-negative bacteria. The most widely distributed of these enzymes is TEM-1, which is encoded by numerous different plasmids (p. 158) and trans-posons (p. 160). This no doubt explains its wide distribution, which includes many enterobacteria, Ps. aeruginosaAcinetobacter spp., Haemophilus influenzae and Neisseria gonorrhoeae. Other types of β-lactamase that are encountered in Gram-negative bacilli include: numerous variants of TEM-1 (some of which may exhibit an altered substrate spectrum); SHV-1 and its many variants (common in Klebsiella spp.); the OXA group of enzymes, which are capable of hydrolysing methicillin and isoxazolylpenicillins; and the PSE group, which hydrolyse carbenicillin at least as fast as benzylpenicillin, and which were thought originally to be confined to Ps. aeruginosa.

Progress has been made towards the control of resistance caused by β-lactamases of Gram-negative organisms by the introduction of ‘β-lactamase-stable’ cephalosporins and the use of β-lactamase inhibitors, such as clavulanic acid. However, novel plasmid-encoded β-lactamases have been described that inactivate even the newer ‘β-lactamase-stable’ β-lactam agents. Many of these novel enzymes seem to be derived by mutation from the widely distributed TEM-1 and SHV-1 β-lactamases.

Most β-lactamases have serine at the active site and are often referred to as serine β-lactamases. However, a group of metallo-β-lactamases that require zinc (and are therefore inhibited by EDTA) are also found in diverse organisms including Acinetobacter spp. and the Bacteroidesgroup. These enzymes hydrolyse virtually all β-lactam compounds, including the carbapenems (p. 24) and enzyme inhibitors such as clavulanic acid do not offer protection. Serine β-lactamases that inactivate carbapenems have also been described. Fortunately, strains that elaborate such enzymes are still relatively uncommon, although they are now being reported with increasing frequency from many different countries.

A simplified classification of the common bacterial β-lactamases is shown in Table 12.2.

Table 12.2 Simplified categorization of the most common bacterial β-lactamases

 

 

 

 

Inhibited by:

 

Group

Molecular class

Preferred substrates

Clavulanic acid

EDTA

Representative enzymes

 

1

C

Cephalosporins

No

No

Gram-negative chromosomal enzymes

2a

A

Penicillins

Yes

No

Staphylococcal β-lactamases

2b

A

Penicillins; cephalosporins

Yes

No

TEM series; SHV series

2c

A

Penicillins;
carbenicillin

Yes

No

PSE series

2d

D

Penicillins;
cloxacillin

Yes

No

OXA series

3

B

Most β-lactam antibiotics

No

Yes

Metalloenzymes (carbapenemases)

 

EDTA, ethylenediaminetetraacetic acid

Adapted from the scheme of Bush K., Jacoby G. A. and Medeiros A. A.: Antimicrobial Agents and Chemotherapy 39 (1995), 1211–33.

Aminoglycosides

Resistance to aminoglycosides results largely from interference with the drug transport mechanism following modification of the antibiotic by one or more of a series of enzymes produced by the resistant bacteria. Such aminoglycoside-modifying enzymes are often plasmid-encoded, but have been associated increasingly with the presence of transposons (p. 160) and integrons (p. 161). They are classified according to the precise type of modification performed and by the site of modification on the aminoglycoside molecule. Over 30 such modifying enzymes and their variants have been identified by biochemical or nucleic acid-based methods, and these can be divided into three main groups: amino-glycoside acetylating enzymes (AAC); nucleotidyltransferase enzymes (ANT); and phosphorylating enzymes (APH).

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The acetylating enzymes, of which there are at least 16 types, catalyse the transfer of acetate from acetyl coenzyme A to an amino group on the amino-glycoside molecule. These enzymes modify only deoxystreptamine-containing aminoglycosides (p. 30) and are, therefore, without effect on streptomycin or spectinomycin. In contrast, aminoglycoside nucleotidyltransferases use ATP or other nucleotides as substrates and attach the nucleotide to exposed hydroxyl groups, while phosphotransferases also modify hydroxyl groups, but by attachment of a phosphate molecule. Examples of the most widely distributed enzymes are listed in Table 12.3.

Table 12.3 Examples of some of the most common aminoglycoside-modifying enzymes and their characteristic substrates

 

 

Typical

Bacterial distribution

Enzyme

substrates

Gram-positive

Gram-negative

 

Acetyltransferases

 

 

 

AAC(3)-I

Gen

-

+

AAC(3)-II

Gen, Tob, Net

-

+

AAC(3)-IV

Gen, Tob, Net

-

+

AAC(2′)

Gen, Tob

-

+

AAC(6′)-I

Tob, Amk, Net, Kan

+

+

AAC(6′)-II

Gen, Tob, Net

-

+

Nucleotidyltransferases

 

 

ANT(4′)

Tob, Amk, Kan, Neo

+

(-)

ANT(2″)

Gen, Tob, Kan

-

+

Phosphotransferases

 

 

APH(3′)-III

Kan, Neo

+

-

APH(3′)-VI

Neo, Kan, Amk

+

+

APH(2″)

Gen, Tob, Kan

+

-

 

(-) indicates that this activity is uncommon.

Amk, amikacin; Gen, gentamicin; Kan, kanamycin; Neo, neomycin; Net, netilmicin; Tob, tobramycin. The figure in brackets indicates the site of modification according to the internationally accepted numbering system for the various parts of the complex aminoglycoside molecule (see Fig.12.2).

It is apparent from Table 12.3 that a variety of patterns of cross-resistance can be shown by bacteria elaborating different enzymes, but the pattern is complicated further because many clinical isolates produce more than one enzyme at any one time. It can therefore be difficult to predict which enzymes are present simply from a consideration of the substrate range. Moreover, sensitivity or resistance to any one agent cannot be predicted reliably from results with another, and sensitivity tests must therefore be performed against the agent that is to be used in treatment.

Figure 12.2 shows the structure of kanamycin A, a typical aminoglycoside, and indicates the various sites at which modification can take place. Clearly, the presence or absence of available amino or hydroxyl groupings will affect the susceptibility to various enzymes, and this is the basis of variability within

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the aminoglycoside group. The steric configuration of the groupings is also important: thus the semi-synthetic aminoglycoside amikacin is, structurally, related closely to kanamycin A, but is much less susceptible to enzymic modification because of a hydroxyaminobutyric acid side chain which alters the steric config-uration of the molecule.

 

Fig. 12.2 Structure of kanamycin A, showing the sites at which enzymic modification can occur.

When the aminoglycoside-modifying enzymes were first described, they were considered to be examples of drug-inactivating enzymes analogous to those responsible for resistance to β-lactam antibiotics and chloramphenicol. However, aminoglycoside-modifying enzymes mediate resistance by modifying only small amounts of antibiotic. They are strategically placed near the inner cytoplasmic membrane where they are accessible to acetyl coenzyme A and ATP. As soon as a few molecules of drug are modified, all further transport of drug into the cell becomes blocked.

Chloramphenicol

Resistance to chloramphenicol in both Gram-positive and Gram-negative bacteria is normally associated with production of an enzyme, chloramphenicol acetyl-transferase, which converts the drug to either the monoacetate or diacetate. The

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acetylated drug is unable to bind to the bacterial ribosome and is therefore without effect on protein synthesis. Several different acetyl transferases have been described. Some appear to be genus- and species-specific; others, usually plasmid or transposon-associated, are more widespread. This variety is a little surprising since chloramphenicol has been used less widely than many other antibiotics because of its rare, but serious, toxic side-effects.

Alteration of the target site

Resistance arising from the selection of rare, pre-existent mutants from within an otherwise sensitive bacterial population has been described for many antibiotics. The mutations usually affect the drug target and often confer high levels of resistance in a single step. The emergence of this type of resistance during therapy is an important cause of treatment failure with certain drugs, including rifampicin, the older quinolones, fusidic acid, and various antituberculosis drugs. Clinically, such resistance can be prevented by using combinations of antibiotics, since the likelihood of independent resistance mutations to two or more unrelated antibiotics appearing simultaneously in the same cell is very small; this strategy has been crucial in antituberculosis therapy.

In contrast, variants exhibiting low levels of resistance to almost any antibiotic can be isolated readily from most bacteria. They usually develop in a stepwise fashion, and may be accompanied by other phenotypic changes (e.g. slower growth rate, colonial variation on solid media, reduced virulence) which become more marked as the degree of resistance increases. It is likely that the shifts in penicillin susceptibility of gonococci and pneumococci that have occurred over the years result from such cumulative changes.

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β-Lactam antibiotics

Although the major mechanism of resistance to β-lactam antibiotics involves enzymic inactivation (see above), mechanisms involving target site modification also occur. Thus, strains of Streptococcus pneumoniae with reduced susceptibility to penicillin exhibit alterations in the target penicillin-binding proteins (PBPs; p. 26) that result in reduced ability to bind penicillin. Similarly, methi-cillin resistance in staphylococci is associated with the synthesis of a modified PBP which exhibits decreased affinity for methicillin and other β-lactam antibiotics. Low-level resistance to penicillin in N. gonorrhoeae has also been associated with alterations to PBPs. Such resistance appears to have developed by rare mutational events and to have become disseminated as a result of considerable antibiotic selection pressure.

Streptomycin

Streptomycin binds to a particular protein, designated S12, in the smaller (30 S) ribosomal subunit in bacteria. A single amino acid change in the structure of this protein can prevent the binding of streptomycin entirely and endow the bacteria with resistance to very high concentrations of the drug. The alteration is very specific, since other aminoglycosides, like gentamicin, are unaffected by the change.

Erythromycin and chloramphenicol

Changes in the proteins of the larger (50 S) ribosomal subunit have been implicated in resistance to chloramphenicol and macrolides such as erythromycin. However, erythromycin resistance in staphylococci and streptococci results more usually from methylation of the 23 S ribosomal RNA subunit by an inducible plasmid-encoded enzyme. Methylation of the ribosomal RNA also renders the bacteria resistant to other macrolides, lincosamides (p. 42), and streptogramins (p. 43) by reducing ribosomal binding of these drugs. However, since erythro-mycin is a specific inducer of the methylating enzyme, bacteria carrying a plasmid encoding this property are resistant to the other drugs only in the presence of erythromycin. This phenomenon is known as dissociated resistance.

Fusidic acid

Fusidic acid inhibits translocation of the growing polypeptide chain. Mutations altering the structure of the protein involved (elongation factor G) result in the cell becoming resistant to the action of this antibiotic.

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Quinolones

Intermediate levels of resistance to quinolones in Gram-negative rods are caused by structural alterations to a subunit of the DNA gyrase target (p. 49). High levels of resistance are associated with additional mutations in the secondary target, topoisomerase IV. In Gram-positive cocci the situation is reversed, since topoisomerase IV is the primary target.

Rifampicin

Resistance to rifampicin is invariably the result of a structural alteration to the β subunit of RNA polymerase (involved in the transcription of DNA to messenger RNA) that reduces its binding affinity for rifampicin.

Glycopeptides

Resistance to vancomycin and teicoplanin in enterococci represents a particularly interesting example of target site alteration. Glycopeptides bind to the D-alanyl-D-alanine terminus of the muramyl pentapeptide of peptidoglycan (p. 12). Enterococci that exhibit high-level resistance to glycopeptides produce two new enzymes, a ligase and a dehydrogenase, with formation of a new depsipeptide terminus, D-alanyl-D-lactate, to the pentapeptide. This substitution allows cell wall synthesis to continue in the presence of the antibiotic.

The mechanism of the low-level resistance to glycopeptide antibiotics that is starting to emerge in Staph. aureus and coagulase-negative staphylococci has not been completely elucidated, but appears to be associated with overproduction of peptidoglycan precursors that require increased amounts of drug to saturate them.

Interference with drug transport and accumulation

In addition to reduced drug accumulation resulting from enzymic modification of aminoglycosides (see above), interference with transport of drugs into the bacterial cell is of proven clinical importance as a cause of resistance to tetracyclines, β-lactam antibiotics, and quinolones.

Tetracyclines

Uptake of tetracyclines into normal cells involves an active transport mechanism that uses energy and results in accumulation of drug inside the cell. Plasmid or transposon-mediated resistance to tetracyclines is common in both Gram-positive and Gram-negative bacteria. Generally, there is complete cross-resistance in that a strain resistant to one tetracycline is resistant to all the others, although exceptions

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may be found with minocycline and some investigational compounds. Resistance is often associated with the synthesis of a membrane protein that mediates rapid efflux of tetracyclines from resistant cells by an active mechanism, so that drug entering the cell is removed almost simultaneously and fails to reach an inhibitory level. Such resistance is normally inducible, and full expression of resistance is obtained only after cells have been exposed to sub-inhibitory concentrations of the drug. There is no evidence for enzymic inactivation of tetracycline in clinical isolates of bacteria, or for modification of the ribosomal target. However, some strains of bacteria produce a cytoplasmic protein which appears to have the function of protecting ribosomes from tetracycline attack.

β-Lactam antibiotics

The outer membranes of Gram-negative bacilli vary greatly in permeability to various penicillins and cephalosporins. Most β-lactam agents reach their targets in Gram-negative bacilli by passing through the water-filled pores (porins) that extend across the outer membrane bilayer. The rate of permeation is governed largely by the physical size of a particular β-lactam molecule in comparison with the size of the porin, but ionic charge also plays a part. In some bacteria, resistance can result from changes to the size or function of the porins, so that passage of the antibiotic is prevented. In a few instances, non-specific changes in cell permeability to β-lactam antibiotics are encoded by genes carried on plas-mids; these changes seem to affect the overall outer membrane structure of the cell and are related directly to plasmid carriage.

Chloramphenicol

A few strains of chloramphenicol-resistant Gram-negative bacilli possess a plasmid that appears to confer the property of impermeability to chloramphenicol upon the host cell.

Quinolones

Strains of Esch. coli have been described in which resistance to quinolones is caused by impermeability associated with a decrease in the amount of the OmpF outer membrane porin protein. Such strains may simultaneously acquire resistance to β-lactam antibiotics and some other agents that gain access through the OmpF porin. Resistance caused by active efflux also occurs in Gram-negative bacilli.

Aminoglycosides

A mechanism of resistance to aminoglycosides, unrelated to enzymic modifica-tion, is associated with membrane alterations that affect active transport of the antibiotic into the cell.

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Metabolic bypass

Most common resistance mechanisms can be accommodated in one or other of the three major groups described already. However, there are two known examples in which a plasmid or transposon provides the cell with an entirely new and drug-resistant enzyme that can bypass the sensitive chromosomal enzyme which is also present unaltered in the cell.

Sulphonamides

Sulphonamides exert their bacteriostatic effect by competitive inhibition of dihy-dropteroate synthetase (p. 46). Sulphonamide-resistant strains of Gram-negative bacilli synthesize an additional dihydropteroate synthetase that is unaffected by sulphonamides. The additional enzyme allows continued functioning of the threatened metabolic pathway in the presence of the drug. At least two such such enzymes are widespread in Gram-negative bacilli throughout the world.

Trimethoprim

Trimethoprim blocks a later step in the same metabolic pathway by inhibiting the dihydrofolate reductase enzymes of susceptible bacteria. Resistant strains synthesize a new, trimethoprim-insensitive, dihydrofolate reductase as well as the normal drug-sensitive chromosomal enzyme. At least 14 groups of trimetho-prim-insusceptible dihydrofolate reductases have been described in Gram-negative bacilli, and a further example is found in multi-resistant isolates of Staph. aureus.

The mechanisms discussed above illustrate the diversity of ways in which microbes can become resistant to the drugs deployed against them. However, any attempt to limit the spread of drug resistance requires not only a knowledge of the mechanisms themselves, but also an understanding of the genetic factors that control their emergence and continued evolution. These factors are the subject of the next chapter.