C H c k oCh Hnv Ch

(11.24) tetroxoprini

Bacteria must synthesize folate because they are unable to absorb preformed folate. PAB, an essential constituent in folate metabolism, and sulphonamides have similar chemical structures.

Diaminopyrimidines structurally dissimilar from folic acid have been known for some time to be antagonists of folic acid. The best known example, trimethoprim (11.22), is a potent inhibitor of dihydrofolate reductase (DHFR), the enzyme responsible for converting dihydrofolate (11.21) to tetrahydrofolate (11.23) in E. coli but not in man. Other antifolate inhibitors of DHFR are described later in Table 11.6, from which it can be seen that tetroxoprim (11.24) also possesses significant antibacterial activity, whereas pyrimethanine is an antimalarial agent and methotrexate is used in cancer chemotherapy.

Since sulphonamides and trimethoprim appear to inhibit sequential changes in tetrahydrofolate synthesis, it was a logical step to use a combination of an appropriate sulphonamide (sulphamethoxazole) with trimethoprim as an antibacterial mixture. Such a combination undoubtedly shows synergism in vitro but not necessarily in vivo. This aspect is discussed more fully in Section 11.6.5.

11.3.6 Conclusions and comments

Mechanisms of antibiotic action are increasingly being understood at the molecular level. This is important for two reasons: first, such drugs have proved to be of considerable importance in elucidating the stages in microbial synthetic processes; secondly, knowledge of how a drug acts should lead logically to the development of more potent, selectively toxic chemotherapeutic agents.

11.4 BACTERIAL RESISTANCE TO CHEMOTHERAPEUTIC

AGENTS

The resistance of bacteria to chemotherapeutic agents has long posed a problem and will continue to do so for the foreseeable future. Much is known about the mechanisms of bacterial resistance, and this section will provide a summary of the available information, because the design of new antibiotics is, to a considerable extent, linked to methods of combating this problem.

Bacterial resistance to antibiotics may be either intrinsic, i.e. a natural property of the organism often associated with cellular impermeability, or be acquired, either by mutation in sensitive cell populations or by resistance transfer from one cell to another. Space does not permit a full discussion of resistance expression at the underlying biochemical level, but a summary is provided in Table 11.3. This table demonstrates that bacteria can present resistance to a chemotherapeutic agent by virtue of many different mechanisms.

Some of these mechanisms, in particular enzyme-mediated resistance (Section 11.4.1), impermeability (11.4.2) and target enzyme affinity (11.6.5) will be considered in this chapter.

11.4.1 Enzyme-mediated resistance

Resistance of bacteria to P-lactam antibiotics may be associated with enzymes termed P-lactamases. Some bacteria are capable of producing enzymes that modify streptomycin or other aminoglycoside antibiotics. A description, therefore, of the various types of enzymes is important in understanding the development and design of antibiotics.

11.4.1.1 fi-lactamases

P-lactamases occur widely in nature. They are produced by various Gram-positive and Gram-negative bacteria. In Gram-positive organisms such as staphylococci and Bacillus species, P-lactamase is an inducible enzyme, with low concentrations of various P-lactam antibiotics acting as appropriate inducers, and is released extracellularly. In contrast, P-lactamases of Gram-negative bacteria are usually constitutive, but are induced by high concentrations of appropriate inducing agents in organisms such as Pseudomonas aeruginosa and Enterobacter cloacae. The P-lactamases of Gram-negative bacteria are

Table 11.3 Expression of resistance to antibiotics.

Expression of

Example(s)

Comments

resistance

Enzymatic

Some ß-lactam

See 11.4.1.1

inactivation

antibiotics

See 11.4.1.3

Chloramphenicol

Enzymatic

Some ß-lactam

See 11.4.1.1

trapping

antibiotics

See 11.4.1.2

Enzymatic

Some

See 11.4.2

modification

aminoglycoside

Reduced ability of cells to

Bacterial

antibiotics

take up drugs

impermeability1

Some ß-lactam

Plasmid-mediated decreased

antibiotics

drug accumulation

Aminoglycoside

Difficulty in entering Gram-

antibiotics

negative cells

Tetracyclines,

chloramphenicol,

fusidic acid

Hydrophobic

antibiotics:

novobiocin,

actinomycin D,

erythromycin,

Antibiotic efflux Tetracyclines

Decreased affinity ß-lactam antibiotics of target enzymes Trimethoprim Sulphonamides

Alteration in binding site

Streptomycin

Erythromycin

Glycopeptides

Energy-dependent efflux of accumulated drugs Altered PSEs/PBPs Altered dihydrofolate reductase

Altered dihydropteroate synthetase

Protein S12 component of 30S

ribosomal subunit determines sensitivity or resistance

Ribosomes from resistant cells have lower affinity, resulting from enzymatic methylation of adenine in 23S rRNA

Acquired ligase produces altered peptidoglycan precursors with lower affinity.

1Depends on chemical nature of drug and on type of organism intracellular, being located in the periplasm situated between the inner and outer membranes, and are less potent enzymes than those produced by Gram-positive bacteria. They may be chromosomally or plasmid-mediated (Section 11.4.3).

The effects of P-lactamases generally on susceptible penicillins and cephalosporins are shown in Figures 11.3 and 11.4 respectively. Susceptible penicillins are converted to the corresponding penicilloic acid (11.25) which is inactive; this results from an opening of

Figure 11.3 Effect of p-lactamases on susceptible penicillins.
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