Hugo W.B., Russel A.D.(ed). Pharmaceutical Microbiology

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site for transcription. The a factor is responsible for recognition of the initiation signal

for transcription and the core enzyme possesses the activity to join the nucleotides in

the sequence specified by the gene. Mammalian genes possess an analogous RNA

polymerase but there are sufficient differences in structure to permit selective inhibition

of the microbial enzyme by the semisynthetic rifamycin antibiotic, rifampicin.

Quinolones

The quinolones selectively inhibit DNA gyrase (topoisomerase II), which is not found

in mammalian cells. The gyrase, a tetramer comprising two A and two B subunits, is a

highly versatile enzyme which is capable of catalysing a variety of changes in DNA

topology. These include introduction of negative supercoiling and removal of positive

supercoiling (relaxation), unknotting, and removal of linked structures (decatenation).

Such activities ensure that the daughter chromosomes produced during replication can

segregate in the cytoplasm prior to cell division. The gyrase binds to the chromosome

at a point where two separate double stranded regions cross (this can be at a supercoiled

region, a knotted or a linked (catenane) region). The A subunits cut both DNA strands

on one chain with a 4 base pair stagger, the other chain is passed through the break

which is then resealed. The B subunits derive energy for the reaction by hydrolysis

of adenosine triphosphate (ATP). The precise details of the interaction are not clear

but it appears that the quinolones bind to the A subunits at exposed single strand ends

of the cut DNA chain. The gyrase is unable to reseal the DNA with the result that

the chromosome in treated cells becomes fragmented. The number of fragments

(approximately 100 per cell) is comparable to the number of supercoils in the

chromosome. The action of the quinolones probably triggers secondary responses in

the cells which are responsible for death. One notable morphological effect of quinolone

treatment of Gram-negative rod-shaped organisms is the formation of filaments. Some

of the quinolones may also act upon topoisomerase IV, which appears to be more

important for chromosome segregation in staphylococci.

Nitroimidazoles (metronidazole) and nitrofurans (nitrofurantoin)

These agents also cause DNA strand breakage but by a direct chemical action rather

than by inhibition of a topoisomerase. Metronidazole is active only against anaerobic

organisms. The nitro group of metronidazole is converted to a nitronate radical by the

low redox potential within cells. The activated metronidazole then attacks the DNA

producing strand breakage. Nitrofurantoin is thought to act in a similar manner.

Rifampicin

The action of rifampicin is upon the /3 subunit of RNA polymerase. Binding of just one

molecule of rifampicin inhibits the initiation stage of transcription in which the first

nucleotide is incorporated in the RNA chain. Once started, transcription itself is not

inhibited. It has been suggested that the structure of rifampicin resembles that of two

adenosine nucleotides in RNA; this may form the basis of the binding of the antibiotic

to the j3 subunit. One problem is the rapid development of resistance in organisms due

Mechanisms of action of antibiotics 175

to alterations in the amino acids comprising one particular region of the /3 subunit.

These changes do not affect the activity of the polymerase but render it insensitive to

rifampicin. The action of rifampicin is specific for the microbial RNA polymerase, the

mammalian version being unaffected.

4.5 5-Fluorocytosine

This antifungal agent inhibits DNA synthesis at the early stages involving production

of the nucleotide, thymidylic acid (dTMP). 5-Fluorocytosine (5-FC) is converted by a

deaminase inside fungi to 5-fluorouracil then to the corresponding nucleoside phosphate,

5-fluorodeoxyuridylic acid (5-F-dUMP). This compound then acts as an inhibitor of

thymidylate synthetase which normally produces dTMP from uridine monophosphate

(dUMP) by addition of a methyl group (supplied by a folate cofactor, section 5.1)

to the 5 position of the uracil ring. As this position is blocked by the fluoro group

5-F-dUMP acts as an inhibitor of the enzyme. 5-FC can be considered as a pro-drug, it

has the value of being taken up by fungi as the pyrimidine base, whereas the active

metabolite produced inside the cells would not be taken up because of its negative

charge. Although 5-FC is an important antifungal agent in treatment of life-threatening

infections, resistance can occur due to active efflux of the drug from the cells before it

can inhibit DNA synthesis.

5 Folate antagonists

5.1 Folate metabolism in microbial and mammalian cells

Folic acid is an important cofactor in all living cells. In the reduced form, tetrahydrofolate

(THF), it functions as a carrier of single carbon fragments which are used in the synthesis

of adenine, guanine, thymine and methionine. One important folate-dependent enzyme

is thymidylate synthetase, which produces dTMP by transfer of the methyl group from

THF to dUMP. In this and other folate-dependent reactions THF is converted to

dihydrofolic acid (DHF), which must be reduced back to THF before it can participate

again as a carbon fragment carrier. The enzyme responsible for the reduction of DHF

to THF is dihydrofolate reductase (DHFR; Fig. 8.5) which uses the nucleotide NADPH

as a cofactor. Bacteria, protozoa and mammalian cells all possess DHFR but there

are sufficient differences in the enzyme structure for inhibitors such as trimethoprim

and pyrimethamine to inhibit the bacterial and protozoal enzymes selectively without

damaging the mammalian form. In the case of protozoa such as the Plasmodium

species responsible for malaria, the DHFR is a double enzyme which also contains the

thymidylate synthetase activity.

There is another fundamental difference between folate utilization in microbial

and mammalian cells. Bacteria and protozoa are unable to take up exogenous folate

and must synthesize it themselves. This is carried out in a series of reactions involving

first the synthesis of dihydropteroic acid from one molecule each of pteridine and p-

aminobenzoic acid (PABA). Glutamic acid is then added to form DHF which is reduced

by DHFR to THF. Mammalian cells do not make their own DHF, instead they take it up

from dietary nutrients and convert it to THF using DHFR.

176 Chapter 8

COO"

Sulphonamides

Sulphonamides are structural analogues of PABA. They competitively inhibit the

incorporation of PABA into dihydropteroic acid and there is some evidence for their

incorporation into false folate analogues which inhibit subsequent metabolism. The

presence of excess PABA will reverse the inhibitory action of sulphonamides, as will

thymine, adenine, guanine and methionine. However, these nutrients are not normally

available at the site of infections for which the sulphonamides are used.

DHFR inhibitors

Trimethoprim is a selective inhibitor of bacterial DHFR. The bacterial enzyme is several

thousand times more sensitive than the mammalian enzyme. Pyrimethamine, likewise,

is a selective inhibitor of plasmodial DHFR. Both are structural analogues of the

dihydropteroic acid portion of the DHF substrate. Crystal structures of the bacterial,

plasmodial and mammalian DHFRs, each containing either bound substrate or the

inhibitors have been determined by X-ray diffraction studies. These show how inhibitors

fit tightly into the active site normally occupied by the DHF substrate, forming a pattern

of strong hydrogen bonds with amino acid residues and water molecules lining the

site. Another DHFR inhibitor is proguanil, a guanidine-containing pro-drug which is

Mechanisms of action of antibiotics 177

metabolized in the liver to cycloguanil, an active selective inhibitor of plasmodial DHFR.

Methotrexate is a potent DHFR inhibitor which has an analogous structure to the whole

DHF molecule, including the glutamate residue. It has no selectivity towards microbial

DHFR and cannot therefore be used to treat infections; however, it is widely used as

an anticancer agent. A derivative of methotrexate which is used for treatment of

Pneumocystis carinii infections in acquired immunodeficiency syndrome (AIDS)

patients is trimetrexate. Although it is very toxic to mammalian cells, simultaneous

administration of leucovorin (formyl-THF or folinic acid) as an alternative source of

folate which cannot be taken up by the organism protects host tissues. DHFR inhibitors

can be used in combination with a sulphonamide to achieve a double interference with

folate metabolism. Suitable combinations with matching pharmacokinetic properties

are sulphamethoxazole and trimethoprim (the antibacterial, cotrimoxazole) and sul-

phadoxine and pyrimethamine (the antimalarial, fansidar).

6 The cytoplasmic membrane

6.1 Composition and susceptibility of membranes to selective disruption

The integrity of the cytoplasmic membrane is vital for the normal functioning of all

cells. Bacterial membranes do not contain sterols and, in this respect differ from

membranes of fungi and mammalian cells. Fungal membranes contain predominantly

ergosterol as the sterol component whereas mammalian cells contain cholesterol. Gram-

negative bacteria contain an additional outer membrane structure which provides

a protective penetration barrier to potentially harmful substances, including many

antibiotics. The outer membrane has an unusual asymmetric structure in which

phospholipids occupy the inner face and the lipopolysaccharide (LPS) occupies the

outer face. The outer membrane is attached to the peptidoglycan by proteins and

lipoproteins. The stability of all membranes is maintained by a combination of non-

covalent interactions between the constituents involving ionic, hydrophobic and

hydrogen bonding. The balance of these interactions can be disturbed by the intrusion

of molecules (membrane-active agents) which destroy the integrity of the membrane,

thereby causing leakage of cytoplasmic contents or impairment of metabolic functions

associated with the membrane. Most membrane-active agents which function in this

way, e.g. the alcohols, quaternary ammonium compounds and bisbiguanides (considered

in Chapter 10), have very poor selectivity. They cannot be used systemically because

of their damaging effects upon mammalian cells; instead they are used as skin anti-

septics, disinfectants and preservatives. A few agents can be used therapeutically: the

polymyxins, which act principally upon the outer membrane of Gram-negative bacteria,

and the antifungal polyenes, imidazoles naftidine.

6.2 Polymyxins

Polymyxin B and polymyxin E (colistin) are used in the treatment of serious Gram-

negative bacterial infections, particularly those caused by Pseudomonas aeruginosa.

They comprise a cyclic peptide containing positively charged groups linked by a

tripeptide to a hydrophobic branched chain fatty acid. They bind tightly to negatively

178 Chapter 8

charged phosphate groups on LPS in the outer membrane of Gram-negative bacteria.

The outer leaflet of the membrane structure is distorted, segments of which are released

and the permeability barrier destroyed. The polymyxins can then penetrate to the

cytoplasmic membrane where they disrupt membrane integrity, causing leakage of

cytoplasmic components. Their detergent-like properties are a key feature of this

membrane-damaging action which is similar to that of quaternary ammonium

compounds. The high affinity of polymyxins for LPS is an advantage in treatment of

P. aeruginosa lung infections where neutralization of the endotoxic action of LPS

released from the organisms reduces inflammation.

Polyenes

Amphotericin B and nystatin are the most commonly used members of this group of

antifungal agents. They derive their action from their strong affinity towards sterols,

particularly ergosterol. The hydrophobic polyene region binds to the hydrophobic sterol

ring system within fungal membranes. In so doing, the hydroxylated portion of the

polyene is pulled into the membrane interior, destabilizing the structure and causing

leakage of cytoplasmic constituents. It is possible that polyene molecules associate

together in the membrane to form aqueous channels. The pattern of leakage is

progressive, with small metal ions such as K

leaking first, followed by larger amino

acids and nucleotides. The internal pH of the cells falls as K

ions are released,

macromolecules are degraded and the cells are killed. The selective antifungal activity

of the polyenes is poor, depending on the higher affinity for ergosterol than cholesterol.

Kidney damage is a major problem when polyenes are used systemically to treat severe

fungal infections. The problem can be reduced but not eliminated by administration of

amphotericin as a lipid complex or liposome.

Imidazoles and triazoles

The azole antifungal drugs act by inhibiting the synthesis of the sterol components

of the fungal membrane. They are inhibitors of one step in the complex pathway

of ergosterol synthesis involving the removal of a methyl group from lanosterol.

The 14a-demethylase enzyme responsible is dependent upon cytochrome P

450

. The

imidazoles and triazoles cause rapid defects in fungal membrane integrity due to reduced

levels of ergosterol, with loss of cytoplasmic constituents leading to similar effects

to the polyenes. The azoles are not entirely specific for fungal ergosterol synthesis

and have some action upon mammalian steroid metabolism, for example they reduce

testosterone synthesis.

Naftidine

This synthetic allylamine derivative inhibits the enzyme squalene epoxidase at an early

stage in fungal sterol biosynthesis. Acting as a structural analogue of squalene, naftidine

causes the accumulation of this unsaturated hydrocarbon, and a decrease in ergosterol

in the fungal cell membrane.

Mechanisms of action of antibiotics 179

7 Further reading

Arthur M., Reynolds P. & Courvalin P. (1996) Glycopeptide resistance in enterococci. Trends Microbiol,

4,401-407.

Barry C.E. & Mdluli K. (1996) Drug sensitivity and environmental adaptation of mycobacterial cell

wall components. Trends Microbiol, 4, 275-281.

Bentley P.H. & Ponsford R. (1993) Recent Advances in the Chemistry of Antiinfective Agents. London:

Royal Society of Chemistry.

Brajtburg J., Powderly W.G., Kobayashi G.S. &Medoff G. (1990) Amphotericin B: current understanding

of mechanism of action. Antimicrob Agents Chemother, 34, 183-188.

Coulson C.J. (1994) Molecular Mechanisms of Drug Action. London: Taylor & Francis.

Franklin J.J. & Snow G.A. (1989) Biochemistry of Antimicrobial Action, 4th edn. London: Chapman &

Hall.

Greenwood D. (1995) Antimicrobial Chemotherapy, 3rd edn. Oxford: Oxford University Press.

Hooper D.C. & Wolfson J.S. (1993) Quinolone Antimicrobial Agents. Washington: American Society

for Microbiology.

Lancini G., Parenti F. & Hall G.G. (1995) Antibiotics: a Multidisciplinary Approach. New York: Plenum

Press.

Nagarajan R. (1991) Antibacterial activities and modes of action of vancomycin and related

glycopeptides. Antimicrob Agents Chemother, 35, 605-609.

Russell A.D. & Chopra I. (1996) Understanding Antibacterial Action and Resistance, 2nd edn. New

York: Ellis Horwood.

Sutcliffe J.A. & Georgopapadakou N.H. (1992) Emerging Targets in Antibacterial and Antifungal

Chemotherapy. New York: Chapman & Hall.

Tipper D.J. (1988) Antibiotic Inhibitors of Bacterial Cell Wall Biosynthesis, 2nd edn. Oxford:

Pergamon Press.

Williams R.A.D., Lambert P.A. & Singleton P. (1996) Antimicrobial Drug Action. Oxford: Bios.

180 Chapter 8

Bacterial resistance to antibiotics

1 Introduction

2 Intrinsic and acquired resistance

2.1 Genetic basis of acquired resistance

2.1.1 Chromosomal mutations

2.1.2 Plasmids

2.1.3 Transposons

3 Biochemical mechanisms of resistance

3.1 Inhibitors of nucleic acid synthesis

3.1.1 Sulphonamides

3.1.2 Trimethoprim

3.1.3 Quinolones

3.1.4 Rifampicin

3.2 Inhibitors of protein synthesis

3.2.1 Aminoglycoside-aminocyclitol group

3.2.2 Tetracyclines

3.2.3 Chloramphenicol

3.2.4 Macrolide, lincosamide and

streptogramin (MLS) antibiotics

Introduction

Bacterial resistance to antibiotics has been recognized since the first drugs were intro-

duced for clinical use. The sulphonamides were introduced in 1935 and approximately

10 years later 20% of clinical isolates of Neisseria gonorrhoeae had become resistant.

Similar increases in sulphonamide resistance were found in streptococci, coliforms

and other bacteria. Penicillin was first used in 1941, when less than 1 % of Staphylococcus

aureus strains were resistant to its action. By 1947,38% of hospital strains had acquired

resistance and currently over 90% of Staph, aureus isolates are resistant to penicillin.

Increasing resistance to antibiotics is a consequence of selective pressure, but the

actual incidence of resistance varies between different bacterial species. For example,

ampicillin resistance in Escherichia coli, presumably under similar selective pressure

as Staph, aureus with penicillin, has remained at a level of 30-40% for many years

with a slow rate of increase. Streptococcus pyogenes, another major pathogen, has

remained susceptible to penicillin since its introduction, with no reports of resistance

in the scientific literature. Equally, it is well recognized that certain bacteria are

unaffected by specific antibiotics. In other words, these bacteria have always been

antibiotic-resistant.

Intrinsic and acquired resistance

Antibiotic resistance is classified into two broad types: intrinsic and acquired.

1 Intrinsic resistance. This suggests that inherent properties of the bacterium are

Bacterial resistance to antibiotics 181

3.2.5 Fusidic acid

3.2.6 Mupirocin

3.3 Inhibitors of peptidoglycan synthesis

3.3.1 /3-Lactams

3.3.2 Glycopeptides

3.3.3 Fosfomycin

3.4 Membrane-active antibiotics

3.4.1 Polymyxins

3.5 Multidrug resistance pumps

3.6 Antibiotics with other resistance

mechanisms

3.6.1 Bacitracin

3.6.2 Antimycobacterial drugs

4 The problem of antibiotic resistance

5 Conclusions and comments

6 References

* See also Chapters 5 and 6.

t Newer quinolones have a broad spectrum of activity against Gram-positive and Gram-negative

bacteria. In general, Gram-negative bacteria tend to be more susceptible.

responsible for preventing antibiotic action (Godfrey & Bryan 1984). This type of

resistance is also termed innate. There are many antibiotics active against Gram-positive

bacteria which have no effect on Gram-negative bacteria and vice versa (Table 9.1).

This intrinsic resistance is thought to be associated with the outer cell layers, such as

the outer membrane, which are absent in Gram-positive cells. The Gram-negative cell

envelope is effectively impermeable, preventing certain antibiotics from reaching their

intracellular targets.

2 Acquired resistance. This occurs when bacteria which were previously susceptible

become resistant, usually, but not always, after exposure to the antibiotic concerned.

Intrinsic resistance is always chromosomally mediated, whereas acquired resistance

may occur by mutations in the chromosome or by the acquisition of genes coding

for resistance from an external source normally via a plasmid or transposon. Both

types are clinically important and can result in treatment failure, although acquired

resistance is more of a threat in the spread of antibiotic resistance (Russell & Chopra

1996).

2.1

Genetic basis of acquired resistance

Three genetic elements are responsible for acquired resistance: chromosomes, plasmids

and transposons (Lewis 1989). Each of these will be considered in turn.

2.7.7

Chromosomal mutations

Resistance to certain antibiotics can arise as a consequence of mutations to chromosomal

genes because of changes in the DNA sequence. Mutations can occur due to single

base pair changes. Transitions involve the substitution of one purine (A or G) for another

and therefore one pyrimidine (C or T) for another. Transversions involve a change

from a pyrimidine to a purine and vice versa. Frameshift mutations occur when one or

182 Chapter 9

Table 9.1 Spectrum

Antibiotic

Penicillin

Fusidic acid

Erythromycin

Vancomycin

Aminoglycosides

Nalidixic acidt

Polymixins

Metronidazole

of activity of some antibacterial antibiotics*

Gram-positive bacteria

Streptococci, staphylococci,

corynebacteria, Clostridia,

Listeria

Staph, aureus

Streptococci, staphylococci,

corynebacteria

Streptococci, staphylococci,

Clostridia

Staph, aureus (gentamicin)

Anaerobes only

Gram-negative bacteria

Anaerobes

Legionella, Campylobacter

Coliforms, pseudomonads

Coliforms

Coliforms, pseudomonads

Anaerobes only

two bases are inserted into the DNA sequence, resulting in an altered reading frame

and therefore an altered gene product.

More extensive changes in the DNA sequence (often referred to as macrolesions)

can also occur. Deletions result in the loss of part of the DNA sequence. Insertions add

extra base pairs to a gene. Transversions occur when a segment of the DNA is reversed

and duplications occur when a segment of the DNA is repeated. Some of these changes

also result in frameshifts.

The molecular basis of acquired chromosomal resistance for specific antibiotics is

discussed later in this chapter.

2.7.2 Plasmids

The bacterial chromosome contains all the genes necessary for the growth and replication

of cells. Many, if not most, bacteria also possess additional circular elements of DNA

which are capable of replicating and transferring independently of the chromosome.

These extrachromosomal genetic elements are known as plasmids and can code for a

number of properties including antibiotic resistance. In a bacterial population under

normal circumstances, it is not necessary for all cells within that population to harbour

plasmids. This has the effect of avoiding the production of unnecessary gene products

unless essential for the survival of the population. Assuming that a subset of the

bacterial population maintains such plasmids, selective pressure following exposure to

an antibiotic will ensure that plasmid-containing, and therefore resistant, cells and their

progeny will survive the antibiotic challenge.

Plasmids have the ability to transfer within and between species and can therefore

be acquired from other bacteria as well as a consequence of cell division. This property

makes plasmid-acquired resistance much more threatening in terms of the spread of

antibiotic resistance than resistance acquired due to chromosomal mutation. Plasmids

also harbour transposons (section 2.1.3), which enhances their ability to transfer

antibiotic resistance genes.

Plasmid transfer normally occurs by conjugation or transduction in vivo. Conjugation

requires cell-to-cell contact and involves the transfer of DNA from a donor cell to a

recipient cell. Plasmids which can mediate their own transfer are termed conjugative

plasmids. Some plasmids which do not possess this property can nevertheless be

transferred if they coexist with a conjugative plasmid. These are known as mobilizable

plasmids. Both Gram-negative and Gram-positive bacteria have the ability to conjugate.

Transduction is a process whereby DNA is transferred by bacteriophages, and plays an

important role in the transfer of antibiotic resistance in Gram-positive bacteria such as

Staph, aureus, Strep, pyogenes and the enterococci. Transduction is generally limited

to organisms of the same species and therefore its role in the transfer of antibiotic

resistance is less significant than conjugation.

A third mechanism of plasmid transfer is by transformation, which is the ability of

certain microorganisms to acquire naked DNA from the environment. This is limited

to certain bacteria, notably Neisseria gonorrhoeae, which is naturally competent to

acquire DNA in this manner. Neisseria gonorrhoeae strains have the ability to recognize

DNA from their own species, and are thus selective in their acquisition of naked DNA

from the environment.

Bacterial resistance to antibiotics 183

2.1.3

Transduction and transformation are generally limited to the same or related species

and are therefore not effective as a means of antibiotic resistance transfer across species

boundaries. However, our knowledge of transformation in nature is limited, and the

significance of this mechanism of gene transfer is unknown.

Transposons

Transposons are mobile genetic elements capable of transferring or transposing

independently from one DNA molecule to another. The DNA molecules may be

chromosomes or plasmids. Transposons are normally flanked by short regions of almost

identical DNA sequence known as repeats. These are termed direct repeats if they lie in

the same direction relative to each other, or inverted repeats if they face in opposite

directions. These repeats are thought to function as recognition sequences for enzymes

involved in transposition (the ability of transposons to transfer and integrate into the

recipient DNA molecule). The central region of the transposon often codes for antibiotic

resistance genes. The ability of transposons to mobilize from one DNA molecule to

another has led to them being referred to as jumping genes. Transposons do not require

homologous regions of DNA in order to integrate into a DNA molecule unlike the

normal recombination process in bacterial cells and are therefore a major cause of the

transfer and spread of antibiotic resistance genes among different bacterial species.

Furthermore, it is possible for bacteria to acquire a series of transposons coding for

different antibiotic resistances by insertion in existing plasmids or the chromosome.

Conjugative transposons which can mediate their own transfer have been described in

the last 10 years. These make the transfer and spread of resistance genes more likely

since they do not depend on insertion into conjugative plasmids for their mobilization.

Known mechanisms of acquired resistance determined by chromosomes, plasmids or

transposons are summarized in Table 9.2.

Biochemical mechanisms of resistance

Many different biochemical mechanisms of antibiotic resistance have been described,

184 Chapter 9

Table 9.2 Genetic determinants of resistance

Chromosomally mediated

resistance only

Methicillin

Quinolones

Rifampicin

Plasmid-mediated

resistance*

Aminoglycoside-

aminocyclitols

/^-Lactams

Tetracyclines

Sulphonamides

Trimethoprim

Chloramphenicol

Erythromycin

Fusidic acid

* Resistance may also be chromosomally mediated.

t Multiple resistance genes may be carried on a transposon.

Transposonst

Single, e.g. ampicillin,

chloramphenicol, tetracycline

Multiple, e.g.

ampicillin + streptomycin +

sulphonamide