Hogg S. Essential microbiology

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358 ANTIMICROBIAL AGENTS

if administered orally, it must not be inactivated by the acid environment of the

stomach, and must be capable of being absorbed by the small intestine.

Antibiotics should not

produce a hypersensitiv-

ity (allergic) reaction in

the host. This is caused by

an extreme response by

the host immune system,

and is not the same as

toxicity.

an antibiotic should not have any signiﬁcant effect on

the resident microﬂora of the host.

it should not be easy for the target pathogen to estab-

lish resistance against an antibiotic.

side-effects such as allergic reactions should be

minimal.

it should be sufﬁciently stable to have a good shelf

life, without special storage considerations.

How do antibiotics work?

All antibiotics have the common property of interfering in some way with a normal,

critical function of the target bacterial cell. The most commonly used antibiotics exert

their effect by one of the following methods:

1 Inhibition of cell wall synthesis (group I)

2 Disruption of cell membranes (group II)

3 Interference with protein synthesis (group III)

4 Interference with nucleic acid synthesis (group IV)

Table 14.2 lists examples of each group. Those antibi-

otics belonging to groups I and III are better able to dis-

criminate between procaryotic and eucaryotic cells, and

consequently show more selective toxicity and a higher

therapeutic index.

Group I: Inhibitors of cell wall synthesis

The therapeutic index

provides a measure of

the selective toxicity

of a chemotherapeu-

tic agent. It is the ra-

tio between the con-

centration at which the

substance causes harm

to its host (toxic dose)

and that at which it

is required to be clin-

ically effective (thera-

peutic dose). It is there-

fore desirable for an

antibiotic to have a high

therapeutic index.

The main group which work in this way are the β-lactam

antibiotics, so-called because they contain a β-lactam

ring in their structure. Included among this group are the

penicillins and the cephalosporins. See also Box 14.4.

You may recall from our discussion of bacterial cell

wall structure in Chapter 3 that an important factor in

the strengthening of the peptidoglycan component of

the bacterial cell walls is the cross-linking of chains by

transpeptidation. It is this process which is acted on by

the β-lactams; they bind irreversibly to the transpep-

tidase enzyme, forming covalent bonds with a serine

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ANTIBIOTICS 359

Table 14.2 Some commonly used antibiotic classes

Inhibitors of cell wall synthesis Penicillins, cephalosporins

Disrupters of cell membranes Polymixins, polyenes

Inhibitors of protein synthesis Streptomycin, tetracyclines

Inhibitors of nucleic acid synthesis Rifamycins

residue within the enzyme’s active site. The cell wall continues to form, but becomes

progressively weaker as more new, unlinked, peptidoglycan is set down. Since bacteria

are generally to be found in a hypotonic environment, as the wall weakens, water enters

the cell, leading to swelling and then lysis.

Penicillins The ﬁrst β-lactam antibiotic to be discovered was benzylpenicillin, or

penicillin-G, whose action is restricted to Gram-positive bacteria, because it is unable

to penetrate the Gram-negative cell wall. It is effective against Gram-positive bacteria

when administered intramuscularly, but cannot be taken by mouth because it is broken

down in the acid conditions of the stomach. Another naturally occurring penicillin,

penicillin-V, represented an advance inasmuch as it is less acid-labile and can therefore

Semisynthetic penicillins

are based on the core

structure of the naturally

occurring molecule, with

the addition of chem-

ically synthesized side

chains.

be taken orally. All the penicillins are based on a core

structure or nucleus called 6-amino-penicillanic acid

(Figure 14.2); extensive research has led to the develop-

ment of many variants of this, the so-called semisynthetic

penicillins. These have attached to their nucleus novel

side chains not encountered in nature, and have over-

come some of the problems inherent in naturally occur-

ring penicillins such as instability and narrow speciﬁcity

(Figure 14.3).

Ampicillin is a semi-synthetic penicillin that has a broader speciﬁcity (Box 14.5) than

Penicillin G; it is appreciably more effective against Gram-negative bacteria such as

Salmonella and E. coli, its hydrophobic nature making it better able to penetrate their

outer membrane. It has the additional beneﬁt of being acid-stable and can therefore be

taken orally.

Another drawback to natural penicillins is that they are susceptible to naturally

occurring bacterial β-lactamases (also called penicillinases), which breaks a bond in the

β-lactam core of the penicillin molecule (Figure 14.4). Sometimes, β-lactam antibiotics

Box 14.4 β-lactam antibiotics have a second mode of action

The β-lactams also act by preventing the natural regulation of enzymes called au-

tolysins. These enzymes function by breaking down peptidoglycan in a controlled

fashion, causing breaks to allow for the addition of new peptidoglycan as the cell

grows, and are normally regulated by naturally occurring inhibitors. The β-lactams

neutralise the activity of these inhibitors, leading to further breakdown of the cell

wall.

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Figure 14.2 Naturally occurring penicillins. (a) Penicillin G (benzylpenicillin); (b) penicillin

V. The dotted outline covers the 6-aminopenicillanic acid nucleus present in all variants of

penicillin. The heavy outline denotes the β-lactam ring

Figure 14.3 Some important semisynthetic penicillins. The shaded square represents the

6-aminopenicillanic acid nucleus common to all penicillins (see Figure 14.2)

360

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ANTIBIOTICS 361

Box 14.5 Broad spectrum or narrow spectrum?

Certain antibiotics, due to the mechanism of their action, are only effective against

a few different pathogens, while others can be used successfully against many

different kinds. They are said to have, respectively, a narrow spectrum and a broad

spectrum of activity.

On the face of it, all things being equal, you would expect your doctor to choose

the antibiotic with the broadest possible spectrum of activity, but this isn’t always

the wisest option. When the cause of an infection isn’t known, it makes sense to

hedge one’s bets and prescribe a broad-spectrum antibiotic (‘whatever it is, this

should sort it!’), but this policy is not without its dangers. The drug is likely to kill off

many of the host’s own resident microflora, which can lead to a superinfection,

and the development of antibiotic-resistant strains is also made more likely. If the

identity of the pathogen is suspected, an appropriate narrow-spectrum drug is to be

preferred.

are taken in combination with a β-lactamase inhibitor such as clavulanic acid. This

binds to the β-lactamase with a high afﬁnity, preventing it from acting on the antibiotic.

Some semisynthetic penicillins such as methicillin and oxacillin are resistant to attack by

the β-lactamases that can render certain bacteria resistant to their naturally occurring

forms.

An anaphylactic shock is

an extreme form of hy-

persensitivity reaction.

Penicillin is not an appropriate treatment for the es-

timated 1–5 per cent of adults who show an allergic re-

action to it; in extreme cases, death from anaphylactic

shock can result.

Cephalosporins The cephalosporins, like the penicillins, have a structure based on a

β-lactam ring (Figure 14.5). They also exert their effect on transpeptidases, but gen-

erally have a broader speciﬁcity and are more resistant to the action of β-lactamases.

Ceftriaxone, for example, is now used in the treatment of gonorrhoeal infections, caused

by penicillin-resistant strains of Neisseria gonorrhoeae. In addition, patients who are

allergic to penicillin are often treated with cephalosporins. Cephalosporins were ﬁrst

Figure 14.4 Action of β-lactamase on penicillin. A number of bacteria, especially staphylo-

cocci, possess the enzyme β-lactamase (penicillinase), which inactivates penicillin by cleavage

of the β-lactam ring at the point marked by the arrow

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362 ANTIMICROBIAL AGENTS

CCCNCR

H HO

COOH

Figure 14.5 Cepahalosporins are based on a nucleus of 7-amino-cephalosoranic acid,

which, like the penicillins, features a β-lactam ring (shown as a square). Note that each

molecule has two variable side chains

isolated in the late 1940s from a marine fungus called Cephalosporium acremonium,

and came into general use in the 1960s. So-called second, third and fourth genera-

tion cephalosporins have been developed to widen the spectrum of activity to include

many Gram-negative organisms, and to keep one step ahead of pathogens developing

resistance to earlier versions.

Both penicillins and cephalosporins are also used prophylactically, that is, in the

prevention of infections, prior to surgery in particularly vulnerable patients.

Other antibiotics that affect the cell wall Carbapenems are β-lactam antibiotics pro-

duced naturally by a species of Streptomyces. A semisynthetic form, imipenem, is active

against a wide range of Gram-positive and -negative bacteria, and is used when resis-

tance to other β-lactams has developed.

Bacitracin and vancomycin are two other antibiotics that exert their effect on the cell

wall, but by a different mechanism. Bacitracin is derived from species of Bacillus and

acts on bactoprenol pyrophosphate, the lipid carrier molecule responsible for transport-

ing units of peptidoglycan across the cell membrane to their site of incorporation into

the cell wall (see Chapter 3). Its use is restricted to topical (surface) application, since

its use internally can cause kidney damage. Vancomycin is a highly toxic antibiotic with

a narrow spectrum of use against Gram-positive organisms such as streptococci and

staphylococci. It is particularly important in its use against infections caused by organ-

isms resistant to methicillin and the cephalosporins, such as methicillin-resistant Staphy-

lococcus aureus (MRSA) (see Resistance to Antibiotics below). It is not absorbed from

the gastrointestinal tract and is therefore most commonly administered intravenously.

Group II: Antibiotics that disrupt cell membranes

Polymixins are a class of antibiotic that act by disrupting the phospholipids of the

cytoplasmic membrane and causing leakage of cell contents. Produced naturally by a

species of Bacillus, polymixins are effective against pseudomonad infections of wounds

and burns, often in combination with bacitracin and neomycin (an inhibitor of protein

synthesis; see below). Their toxicity makes them unsuitable for internal use. Polyene

antibiotics such as amphotericin and nystatin are antifungal agents that act on the

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ANTIBIOTICS 363

sterol components of membranes; they are discussed more fully towards the end of this

chapter.

Group III: Inhibitors of protein synthesis

Antibiotics that act by affecting protein synthesis generally have a relatively broad spec-

trum of action. As we saw in our historical review earlier in this chapter, streptomycin

was the ﬁrst antibiotic that was shown to be effective against Gram-negative organisms.

Its discovery in 1943 was particularly welcome since such organisms were unaffected

by penicillin or sulphonamides. It proved to be particularly useful in the treatment of

tuberculosis, the causative agent of which, Mycobacterium tuberculosis, is protected

against the effects of penicillin by the waxy layer of mycolic acids in its cell wall.

Streptomycin belongs to a group of antibiotics called aminoglycosides, which act

by binding to the 30S subunit of the bacterial ribosome, preventing attachment of

the 50S subunit to the initiation complex (Figure 14.6). They can thus discriminate

between procaryotic (70S) and eucaryotic (80S) ribosomes, and consequently have a

relatively high therapeutic index (although not as high as cell wall inhibitors). Other

members of this group are gentamicin, kanamycin and neomycin. Like some other

‘wonder drugs’, streptomycin has proved to have undesirable side-effects; these have

led to it being replaced in most applications by safer alternatives. In addition, bacterial

resistance to streptomycin is widespread, further diminishing its usefulness. Use of the

5’

PE A

3’

50S

subunit

30S

subunit

tRNA

mRNA

Figure 14.6 Inhibitors of protein synthesis. (a) By binding to the 30S subunit of the bac-

terial ribosome, aminoglycosides block the attachment of the 50S subunit. This prevents

completion of the initiation complex, thus protein synthesis is inhibited. (b) Tetracyclines

distort the shape of the 30S subunit, preventing the attachment of the appropriate amino-

acyl tRNA. (c) Chloramphenicol inhibits peptidyltransferase and prevents formation of new

peptide bonds. (d) Macrolides such as erythromycin bind to the 50S subunit, preventing

elongation of the growing peptide chain

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364 ANTIMICROBIAL AGENTS

aminoglycosides as a group has diminished since the development of later generation

cephalosporins and the tetracyclines.

Tetracyclines also work by binding to the 30S ribosomal subunit, preventing the

attachment of aminoacyl tRNA, and therefore extension of the peptide chain (Fig-

ure 14.6). They are yet another group of antibiotics produced by Streptomyces spp.

Both natural and semisynthetic tetracyclines are easily absorbed from the intestine,

allowing them to be taken orally. Coupled with their broad speciﬁcity (the broadest of

any antibiotic), this led to inappropriately widespread use in the years following their

discovery, sometimes resulting in complications caused by the destruction of the normal

resident microﬂora. Tetracyclines are still used for a number of applications, notably to

treat a variety of sexually transmitted diseases.

Two important antibiotics which act on the larger, 50S, subunit of the procaryotic

ribosome are erythromycin and chloramphenicol. Both combine with the subunit in

such a way as to prevent the assembly of amino acids into a chain (Figure 14.6). Chlo-

ramphenicol was the ﬁrst antibiotic to be discovered with a broad spectrum of activity;

it also derives originally from Streptomyces spp., but is nowadays produced synthet-

ically. Its use has become severely restricted since it was shown to have some serious

side-effects, notably on the bone marrow, but it remains the agent of choice for the

treatment of typhoid fever.

Erythromycin is the best known of the macrolide group of antibiotics. Unlike chlo-

ramphenicol, it has a large hydrophobic molecule and is unable to gain access to most

Gram-negative bacteria, thus restricting its spectrum of activity. Erythromycin can be

taken orally and has a similar spectrum of activity to penicillin G; it is often used instead

of penicillin in the treatment of staphylococcal and streptococcal infections in children.

It is particularly appropriate for this application as it is one of the least toxic of all

commonly used antibiotics.

Group IV: Inhibitors of nucleic acid synthesis

Rifampin belongs to a group of agents called rifamycins. It acts by inhibiting the enzyme

RNA polymerase, thereby preventing the production of mRNA. Rifampin is used against

the mycobacteria that cause tuberculosis, an application for which its ability to penetrate

tissues makes it well suited. Unlike most antibiotics, rifampin interacts with other drugs,

often reducing or nullifying their effect. When used in high doses, it has the unusual side-

effect of turning secretions such as tears, sweat and saliva, as well as the skin, an orange-

red colour. As we have already seen, the quinolone group of synthetic antimicrobial

drugs act by disrupting DNA replication.

Resistance to antibiotics

The global increase in resistance to antimicrobial drugs, including the emergence of bacterial

strains that are resistant to all available antibacterial agents, has created a public health problem

of potentially crisis proportions.

American Medical Association, 1995

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RESISTANCE TO ANTIBIOTICS 365

Box 14.6 Where did antibiotic resistance come from?

Have genes responsible for antibiotic resistance always existed in nature, or have

they arisen since the development and widespread use of antibiotics? The answer,

almost certainly, is the former. A sample of an E. coli strain, freeze-dried in 1946, was

revived many years later, and found to have plasmid-encoded genes for resistance

to streptomycin and tetracycline, neither of which were in clinical use until some

years after the culture was preserved. It seems likely that bacteria possessed these

genes to protect against naturally occurring antibiotics, an idea supported by the

fact that R-plasmids have been found in non-pathogenic soil bacteria. Also, resis-

tance to a number of antibiotics has been demonstrated in soil and water bacteria

from sources sufficiently remote to be free from anthropogenic influence.

As we have already suggested, the impact of certain antibiotics can be greatly reduced

due to the development of resistance by target pathogens (Box 14.6). This represents the

greatest single challenge facing us in the ﬁght against infectious diseases at the start of

the 21st century. Fleming himself foresaw that the usefulness of penicillin might become

limited if resistant forms of pathogens arose.

Nosocomial infections

are ones that are ac-

quired in hospitals or sim-

ilar locations. Some 5--10

percent of hospital pa-

tients acquire such an in-

fection during their stay.

This may prove to be

fatal, especially among

the elderly and immuno-

compromised. As well as

the human cost, such in-

fections extend the av-

erage time spent in hos-

pital and therefore add

greatly to the costs of

treatment.

Not long after penicillin was put into general use,

strains of Staphylococcus aureus were found which

did not respond to treatment, and by 1950 penicillin-

resistant S. aureus was a common cause of infections in

hospitals. A decade later, a semi-synthetic form of peni-

cillin, methicillin, was introduced; this was not affected

by the β-lactamase enzymes that inactivated Penicillin

G, and was used to treat resistant forms. Within years,

however, came the ﬁrst reports of strains of S. aureus

that did not respond to methicillin. The incidence of

methicillin-resistant S. aureus (MRSA) has increased

greatly since, and it represents the major source of noso-

comial infections. In 1980, synthetic ﬂuoroquinolones

were introduced to counter the threat of MRSA, but

within a year 80 per cent of isolated strains had devel-

oped resistance to these too. Vancomycin is regarded as a

last-resort treatment for MRSA, for a number of reasons;

it has a number of serious side-effects, its widespread use

would encourage resistance against it, and it is extremely

expensive.

A case of vancomycin-resistant Staphylococcus aureus (VRSA) emerged in Japan in

1996; a few months later it had reached the USA. This represents a serious threat; some

of these strains respond to treatment with a cocktail of antibiotics, but already people

have died from untreatable VRSA infections. In 2003, a strain of VRSA was shown

to have obtained its vancomycin resistance by cross-species transfer from a strain of

Enterococcus faecalis.

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366 ANTIMICROBIAL AGENTS

How does antibiotic resistance work?

We saw earlier in this chapter how antibiotics exert their effects in a variety of ways, so

it should come as no surprise that there is no single mechanism of resistance. Resistance

may be natural, that is, intrinsic to the microorganism in question, or it may be acquired.

Some bacteria are able to resist antibiotic action by denying it entry to the cell;

penicillin G for example is unable to penetrate the Gram negative cell wall. Others

can pump the antibiotic back out of the cell before it has had a chance to act, by

means of enzymes called translocases; this is fairly non-speciﬁc, leading to multiple drug

resistance. Other bacteria are naturally resistant to a particular antibiotic because they

lack the target for its action, for example, mycoplasma do not possess peptidoglycan,

the target for penicillin’s action.

To avoid the action of an antibiotic, bacteria may be able to use or develop alter-

native biochemical pathways, so that its effect is cancelled out. Many pathogens can

secrete enzymes that modify or degrade antibiotics, causing them to lose their activ-

ity; we have already seen that penicillins can be inactivated by enzymatic cleavage of

their β-lactam ring. Similarly, chloramphenicol can be acetylated, while members of the

aminoglycoside family can be acetylated, adenylated or phosphorylated, all leading to

loss of antimicrobial activity.

Mutations may occur which modify bacterial proteins in such a way that they are

not affected by antimicrobial agents. You will recall that streptomycin normally acts by

binding to part of the 30S subunit on the bacterial ribosome; the actual binding site is

a protein called S12. Mutant forms of the S12 gene can lead to a product which still

functions in protein synthesis, but loses its ability to bind to streptomycin. Similarly,

mutations in transpeptidase genes in staphylococci means they do not bind to penicillin

any more, so cross-linking of the cell wall is not inhibited.

How does resistance arise?

Occasionally, mutations occur spontaneously in bacteria, which render them resistant

to one antibiotic or another. Usually the mutation leads to a change in a receptor or

binding site such as those just described, rendering the antibiotic ineffective. The changes

are usually brought about by point mutations (see Chapter 11) occurring at very low

frequency on chromosomal DNA. Bacteria can, however, become resistant much more

rapidly by acquiring the mutant resistance-causing gene from another bacterium. This

is called transmissable antibiotic resistance; it occurs mainly as a result of bacterial

conjugation, and is the cause of most of the resistance problems we presently face.

Transmissable resistance was ﬁrst reported in Japan in the late 1950s, when multi-drug

resistance in Shigella was shown to have been acquired by conjugation with resistant

E. coli in a patient’s large intestine. E. coli is known to transfer R (resistance) plasmids

to several other gut bacteria including Klebsiella, Salmonella and Enterobacter, as well

as Shigella. Whereas chromosomal mutations usually result in a modiﬁcation to the

drug’s binding site, genes carried on plasmids code for enzymes which inactivate it, (e.g.

β-lactamases) or lead to its exclusion from the cell (translocases).

There is a strong link between the use of a particular antibiotic in a locality and the

incidence of resistant bacterial strains. This is because of selective pressure favouring

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ANTIBIOTIC SUSCEPTIBILITY TESTING 367

the resistant forms of a bacterium. Fortunately this can, at least in part, be reversed,

as several studies have shown, where a more restricted use of certain antibiotics over

several years was followed by a reduction in the incidence of resistant bacterial forms.

Antibiotic susceptibility testing

In order to determine the most appropriate antimicrobial agent to use against an infec-

tion, it is necessary to determine the susceptibility of the pathogen. There are several

ways of doing this, but here we describe the two most commonly employed techniques.

The tube dilution assay determines the minimum inhibitory concentration (MIC) of

the antibiotic, that is, the lowest concentration at which it prevents growth of a given

organism. A series of tubes containing increasingly dilute preparations of the antibiotic

is introduced into a broth with a standard number of test organisms and incubated. The

lowest concentration in the series to show no microbial growth is the MIC (Figure 14.7).

9.9ml

growth

medium

Antimicrobial Agent

0.1ml 0.1ml 0.1ml0.1ml0.1ml0.1ml0.1ml0.1ml

MIC

No growth Growth

Figure 14.7 Minimal inhibitory concentration (MIC). The test organism is incubated with

serially diluted antibiotic. The lowest concentration capable of preventing microbial growth

is the MIC