ANTIBIOTICS

‘ANTIBIOTIC’

A poor definition: "A substance produced by one micro-organism that inhibits other micro-organisms."
This would cover, e.g. carbon dioxide, organic acids, ammonia. These are NOT antibiotics.

A good definition: "A substance produced by a micro-organism, or a similar substance (produced wholly or partly by chemical synthesis) that inhibits the growth of other microorganisms (generally bacteria) at low concentrations."

Antibiotics either kill micro-organisms or inhibit them, e.g. bactericidal agents kill bacteria; bacteriostatic agents inhibit the growth of bacteria.

‘CHEMOTHERAPEUTIC AGENT’

Definition: A chemical used to treat or cure disease.

More particularly, a substance that interferes with the proliferation of cells (microorganisms and other cells, e.g. cancer) at concentrations tolerable to the host (patient).

Anti-microbial chemotherapeutic agents can be anti-bacterial (bactericidal or bacteriostatic), anti-fungal (fungistatic or fungicidal), anti-viral, or anti-protozoal.

All antibiotics are chemotherapeutic agents (used to treat bacterial infections), but not all chemotherapeutic agents are antibiotics!

CONCEPT OF ‘SELECTIVE TOXICITY’

Paul Ehrlich ("The Father of Chemotherapy") first suggested the concept of using chemicals (toxins) to target infectious micro-organisms without harming the patient, i.e. selective toxicity.

Paul Ehrlich

More on Orwell and streptomycin

Aminoglycosides can be ototoxic (affect hearing) and nephrotoxic (affect kidney nephrons). Therefore the levels of these drugs have to be monitored to ensure they do not rise above a certain level which would be toxic to the patient. For example, gentamicin levels in blood should not rise above 10 µg per ml.

This makes aminoglycosides far from 'perfect' but, in addition to TB therapy, they may be used in life-threatening situations where other antibiotics are ineffective, e.g. use of gentamicin in undiagnosed meningitis.

3. TETRACYCLINES

These are obtained from various species of streptomycete bacteria but some are now semi-synthetic.

Consist of four hexagonal rings linked together and three varying R groups.

Tetracycline

e.g.     oxytetracycline,
            chlortetracycline,
            minocycline,
            doxycycline.

Tetracyclines are broad spectrum antibiotics (have activity against a range of G+ and G- bacteria). Doxycycline can also be used against the protozoa causing malaria (Plasmodium spp.).

However, broad-spectrum activity encourages resistance to develop and this has certainly happened with some bacteria in response to exposure to tetracyclines. Broad-spectrum antibiotics are "the last refuge of the diagnostically destitute", i.e. if a doctor is unsure what is causing an infection, they may opt for a broad-spectrum antibiotic as a safeguard. But, if the identity of the infectious agent is known, it is generally best to prescribe a narrow-spectrum antibiotic which is less likely to encourage resistance in other species of bacteria. Having said that, a broad-spectrum antibiotic may be used as a prophylactic to prevent disease in patients particularly susceptible to infection, e.g. those on immuno-suppressive drugs or those immuno-compromised by another disease.

Contra-indications: Tetracyclines discolour developing bones and teeth making them brown. Therefore, they should only be given to adults and not to infants, children or pregnant women.

4. RIFAMYCINS

Have a complex molecular structure of many rings.

Rifamycin

First isolated in 1957 from the bacterium, Amycolatopsis mediterranei (Nocardia mediterranei ).

Some have activity against Mycobacterium tuberculosis, Mycobacterium leprae and AIDS-related mycobacterialinfections.

e.g.      rifampicin,
            rifabutin,
   rifapentine

5. MACROLIDES

Contain large lactone rings linked with amino sugars.

e.g. erythromycin

Erythromycin

Erythromycin is red, hence its name ('erythro-' is Greek for red).

It has activity against most G+ bacteria but also Neisseria spp., Haemophilus spp., and Legionella pneumophila (the cause of Legionnaires disease - an atypical pneumonia). The treatment for a typical pneumonia such as one caused by Streptococcus pneumoniae (the pneumococcus) is usually a penicillin. Penicillins do not work against Legionella pneumophila and, therefore, it is important that an atypical pneumonia is recognized quickly and the correct antibiotic administered or the patient may die.

6. POLYPEPTIDE ANTIBIOTICS

A diverse group.

Bacitracins (mainly active against G+ bacteria but high toxicity).
Polymyxins (from Bacillus polymyxa and active against many G- bacteria).
Capreomycin and viomycin (have anti-TB activity).
Vancomycin (from Streptomyces orientales)

Vancomycin

Vancomycin may be used against Enterococcus spp. and strains of Staphylococcus aureus that are resistant to meticillin (methicillin) and other penicillins (MRSA). However, resistance to vancomycin has developed in some strains.

7. CHLORAMPHENICOL

This compound cannot be classified in any of the previous chemical classes.

Originally derived from a streptomycete but now chemically synthesized.

Chloramphenicol

Has broad-spectrum activity.

Has anti-rickettsial activity (e.g. treatment of typhus and Rocky Mountain spotted fever).

Used to treat typhoid fever (caused by Salmonella typhi) and often reserved for this purpose to reduce the risk of resistance developing.

8. SYNTHETIC ANTIBACTERIAL AGENTS

These are not produced by micro-organisms and, therefore, do not fit the strict definition of an antibiotic, viz.

"A substance produced by a micro-organism, or a similar substance (produced wholly or partly by chemical synthesis) that inhibits the growth of other microorganisms at low concentrations."

Sulphonamides

Derived from dyes (1935).

Active against a range of bacteria but nowadays mainly used for urinary tract infections (UTIs) because they are excreted rapidly from the body via the kidneys and urinary tract and, therefore, accumulate in the infected part of the body.

Quinolones

Derivatives of nalidixic acid.

Nalidixic acid

Active against several G- bacteria. Used for UTIs but may be neurotoxic.

Quinolines

e.g. 'EnteroVioform' (a 8-hydroxyquinoline derivative) was sold as a prophylactic for travellers' diarrhoea until it was discovered that it could cause blindness due to its toxicity for the optic nerve, particularly in people of Japanese origin or descent.

Nitrofuran compounds

Hundreds have been synthesized but only a few are used clinically.

e.g. nitrofurantoin (for treatment of UTIs)

e.g. furazolidone (for treatment of diarrhoea)

Metranidazole

Originally developed as an anti-protozoal drug ('Flagyl') for the treatment of, for example, Trichomonas vaginalis infections, it was later found that it also had activity against anaerobic bacteria such as Bacteroides species.

Metranidazole

ANTI-TUBERCULAR DRUGS

Treatment of tuberculosis is often difficult because the bacteria can develop resistance to many drugs used. Resistance problems are compounded by the fact that the disease is chronic and can last for months, or years, thus giving the bacteria more opportunity to develop resistance. The slow growth rate also is a factor in resistance since the low metabolic rate of Mycobacterium tuberculosis leaves it less prone to inhibition by drugs.

For this reason TB is invariably treated using a combination or 'cocktail' of drugs so that, if resistance develops to one, hopefully it may not to the others.

The standard drugs used are called "first-line" and are usually used in various combinations of the two main ones:

isoniazid (an unusual drug that only affects TB) INH
a rifamycin (rifampicin, rifampin) RIF

Plus 2 or 3 from:

streptomycin SM
pyrazinamide PZA
ethambutol EMB

Isoniazid

However, resistance can develop, particularly to streptomycin and other first-line drugs.

If front-line drugs fail due to resistance, then second-line drugs are used but some require monitoring and some have particularly bad side effects, especially in long-term use. Second-line drugs include:

amikacin
capreomycin
ciprofloxacin
clofazimine
cycloserine
ethionamide
kanamycin
ofloxacin
para-aminosalicyclic acid (PAS)

PAS

Some strains of the bacterium in certain parts of the world have developed resistance to most, if not all, drugs (multi-drug resistant tuberculosis - MDR-TB). This means that there is nothing left to treat victims and they will die.

More on anti-tubercular drugs 03

ANTI-FUNGAL DRUGS

Some fungi (yeasts and moulds) can be pathogenic, e.g. Candida albicans causing thrush, and dermatophytes causing athlete's foot, ringworm, barber's itch, jockey's crutch.

However, fungi are eukaryotic, like the patient, and there are fewer differences and target sites to exploit compared to prokaryotic pathogens. This means that there are comparatively few anti-fungal agents available.

Griseofulvin for dermatophyte infections.

Griseofulvin

Nystatin and amphotericin B for treatment of thrush (caused by Candida albicans).

Nystatin

ANTI-PROTOZOAL DRUGS

There are drugs which exploit the differences between a protozoal pathogen such as Plasmodium (malaria) and the patient. However, as for fungi, the protozoan and the patient are both eukaryotic and, therefore, there are fewer differences and target sites compared to prokaryotic pathogens. This means that there are comparatively few anti-protozoal agents despite the need for more due to resistance problems in some malaria strains in certain parts of the world.

Quinine for malaria.

Quinine

Chloroquine for malaria and amoebiasis.
Metronidazole for Trichomonas vaginalis infection and amoebic dysentery (also used to treat infections caused by anaerobic bacteria).

Metronidazole

ANTI-VIRAL DRUGS

Since viruses are non-cellular and integrate completely with their host cells, there is a lack of 'selective toxicity' and very few target sites to exploit. Of the relatively few anti-viral drugs available, some are analogues of nucleic acid (DNA or RNA) bases and inhibit the virus by becoming incorporated into the viral genome and interfering with the reading of the genetic code.

Amantadine hydrochloride for influenza A virus

Amantadine hydrochloride

Methisazone for Variola (smallpox)

AZT (3′-Azido-3′-deoxythymidine) for HIV

AZT

Acyclovir ('Zovirax') for Herpes simplex

Acyclovir

Interferons

CLASSIFICATION OF ANTIBIOTICS BY TARGET SITE

Remember that an antibiotic is used to exploit a difference between the prokaryotic pathogen and the eukaryotic patient. A target site is a structure or biochemical pathway that is affected or inhibited by the antibiotic but is absent in the patient or different in some way from that in the pathogen. This is an application of Ehrlich's concept of 'selective toxicity'; the antibiotic being a 'magic bullet'.

The target site and the mode of action of an antibiotic are determined, not surprisingly, by its chemical structure.

There are 5 target sites for antibiotics in the bacterial cell:

1. CELL WALL SYNTHESIS

The Gram-positive cell wall contains:

The Gram-negative cell wall contains:

The higher lipid content and the membranous nature of the G- cell wall account to some extent for the generally greater resistance of G- bacteria to antibiotics compared to G+ bacteria. In addition, the smaller proportion of peptidoglycan in the G- cell wall means that they respond less well to antibiotics affecting peptidoglycan synthesis such as the β-lactams.

Peptidoglycan is unique to prokaryotic cells and its synthesis makes an ideal target site since its inhibition will not affect the patient who has no peptidoglycan and, in fact, no cell wall at all!

Also, peptidoglycan is synthesized continuously by the growing bacterial cell and, therefore, the target site is always vulnerable to inhibition by some antibiotics.

Linear polymers

Stage 4. Cross-linking of the linear polymer molecules then occurs to give rigidity and strength to the cell wall.

Cross-linked linear polymers

Several different enzymes are involved at each of the four main stages. Inhibition of any of the stages will affect the quantity and quality of the peptidoglycan product and the final cell wall structure.

Antibiotics are available that operate on the four different stages of peptidoglycan synthesis.

Cycloserine inhibits Stage 1 (pentapeptide formation).
Vancomycin inhibits Stage 2 (pentapeptide binding).
Bacitracin inhibits Stage 3 (linear polymerization) and also affects teichoic acid synthesis in G+ bacteria.
β-lactam antibiotics such as penicillins and cephalosporins inhibit Stage 4 (cross linking).

The β-lactam antibiotics inhibiting Stage 4 are the most important in terms of the number of drugs available and degree of use.

Mode of action

β-lactam antibiotics have a similar structure to the D-alanyl-D-alanine end of the peptidoglycan molecule. They bind to the enzyme responsible for transpeptidation and formation of the cross links. This binding is reversible. There is competitive inhibition of the enzyme whose active sites are occupied by the antibiotic and therefore unavailable to the 'real' substrate, peptidoglycan. The extent of the effect of the antibiotic will depend on its relative concentration compared to the substrate. The result will be that some cross links are missing from the final peptidoglycan molecule and the cell wall will be weaker than normal. If the cell wall is weakened sufficiently, the bacterial cell will lyse and die due to the influx of water by osmosis (assuming the cell is in a hypotonic environment).

Peptidoglycan weakened by missing cross links

Bacterial cells in an isotonic or hypertonic environment will not lyse and may survive the antibiotic treatment. This is rare but can occur in some parts of the body such as the urinary and respiratory tracts. Such bacteria may then regain their cell wall once use of the antibiotic has ceased and the patient will suffer a relapse of the infection.

Penicillin-binding proteins (PBPs) are also important in the process.

2. PROTEIN SYNTHESIS

Both prokaryotic (bacterial) and eukaryotic (human) cells synthesize protein using ribosomes but some of the details of the processes (transcription, translation) are different, especially the type of ribosome - the eukaryotic type is approximately twice the size of the prokaryotic type (80S compared to 70S). This is one of the differences that offer a target site for some antibiotics. Some antibiotics will affect the 70S ribosome, or one of its sub-units, but not the 80S ribosome. However, note that animal (and plant cells) do contain 70s-type ribosomes in their mitochondria (and chloroplasts) but these organelles are surrounded by a double membrane which will prevent some antibiotics entering. Also, an antibiotic used for a relatively short time for an acute infection is unlikely to have much long-term effect on the patient's mitochondrial ribosomes.

Incidentally, the existence of 70S-type, i.e. bacterial-like, ribosomes in animal cells and plant cells is evidence that mitochondria and chloroplasts evolved from symbiotic bacteria. Another piece of evidence is that both organelles have their own DNA in the form of a circle - just like bacteria.

Ribosomes (S = sedimentation coefficient)

Chloramphenicol affects translation with 70S ribosomes by inhibiting the enzyme causing transfer of peptide to the new amino acid at the acceptor site.
Erythromycin affects transcription by binding to the 50S sub-unit (only found in 70S ribosomes) and interfering with the translocation of ribosomes along the messenger RNA (mRNA).
Streptomycin affects both transcription and translation by inhibiting the binding of transfer (tRNA) and messenger RNA (mRNA) to the 30S sub-unit (only found in 70S ribosomes).

3. NUCLEIC ACID SYNTHESIS

The growth and division of any cell depends on the synthesis of DNA and the synthesis of tRNA and mRNA.

However, although the patient's cells also synthesize nucleic acids, there are differences from the processes in bacteria which can be exploited in anti-bacterial chemotherapy.

Nalidixic acid and the quinolones work on G- bacteria by inhibiting the enzyme gyrase which is only found in bacteria and responsible for the supercoiling of DNA. Supercoiling is part of the bacterial DNA packaging mechanism. Eukaryotic cells package their DNA in a completely different way using histone proteins.
Rifampicin inhibits bacterial RNA polymerase but does not affect eukaryotic RNA polymerase which is different. No RNA means no protein synthesis means no cell growth.

4. CELL MEMBRANE PERMEABILITY

Few compounds are selective enough to target the pathogen's cell membranes and not the patient's ones. However, there are some examples of drugs which cause the pathogen's cell membrane to become more permeable ('leaky') and allow the escape of essential nutrients, co-factors, etc. thus inhibiting cell growth.

Polymyxins affect the permeability of G- bacterial cell membranes (but high toxicity for patient).
Amphotericin B and nystatin affect the permeability of fungal cell membranes, e.g. Candida albicans.

5. FOLATE SYNTHESIS

Folate (folic acid) is essential for all living cells and eventually feeds through into the synthesis of nucleic acids (DNA and RNA). Animals and humans are unable to synthesize folate and obtain it from their diet. But many bacteria synthesize their own folate. Therefore any drug that interferes with folate synthesis will inhibit the growth of these bacteria. To understand how this can work you need to know that such bacteria are impermeable to folate and cannot absorb it from their surroundings (otherwise a pathogenic bacterium would simply take folate from the patient's blood).

Examples:

Sulphonamides interfere with the reaction of PABA (p-aminobenzoate) an intermediate in bacterial folate synthesis.
Trimethoprim is an analogue of folate and inhibits the final synthesis enzyme. This drug can be used in combination with a sulphonamide in the treatment of UTI.

ANTIBIOTIC COMBINATIONS

Sometime more than one antibiotic is used to treat an infection. This may be because the organism is likely to develop resistance to one of them but less likely to develop resistance to all. This is the principle behind using 'cocktails' of anti-microbial drugs to treat tuberculosis (See anti-tubercular drugs). Also, sometimes antibiotic combinations are used prophylactically to prevent infection in immuno-compromised patients.

However, some antibiotics work better in combination than the sum of their parts - a phenomenon known as synergism. Most antibiotics work independently of each other and the effect is known as additivity. The effect to definitely avoid is antagonism when two or more antibiotics negate each other's effect and it would have been better to use one antibiotic alone rather than a combination. An effect somewhere between additivity and antagonism is known as indifference. Whether we get synergism, additivity, indifference or antagonism depends on the antibiotic combination, the species of bacterium, and the strain.

A simple way of representing the results of antibiotic combinations is:

Combination effect	Result
Synergism	1 + 1 = >2
Additivity	1 + 1 = 2
Indifference	1 + 1 = >1, <2
Antagonism	1 + 1 = <1 (possibly zero)

Examples:

Effect	Antibiotic combination	Bacterium
Synergism	Carbenicillin + gentamicin	*Enterococcus faecalis*
	Rifampicin + clindamycin	*Bacillus anthracis*
	Trimethoprim + sulphamethoxazole	*Escherichia coli*
Additivity	Most	Most
Indifference	Ciprofloxacin + penicillin	*Bacillus anthracis*
Indifference	Erythromycin + penicillin	*Streptococcus pneumoniae*
Antagonism	Penicillin + tetracycline	*Bacillus anthracis*
Antagonism	Vancomycin + clindamycin	Staphylococcus aureus

Note: the performance of antibiotics, both in combinations and alone, may vary between in vitro and in vivo use. Therefore, it is important to not always put too much emphasis on laboratory in vitro test results because the effect on an infectious agent in a patient (in vivo) may be different.