Antimicrobials 1: Origins and modes of action

Dr Fiona Walsh

Objectives of lecture

• Antibiotic discovery

• Time-line of currently prescribed antibiotics

• General principles of antimicrobial agents

• How antibiotics inhibit or kill bacteria

• Introduction to all antibiotic classes

Definitions

• Antibiotic is a naturally occurring substance that inhibits or kills bacteria

• Antibacterial is a natural, semi-synthetic or synthetic substance that inhibits bacteria

• Antimicrobial agent is a natural, semi-synthetic or synthetic substance that inhibits microbes

Antibiotic discovery

19th CenturyLouis Pasteur Identified bacteria as causative agent of

Robert Koch disease. (Germ theory)

Now know what is causing disease, need to find out how to stop it.

1877 Pasteur Soil bacteria injected into animals made Anthrax harmless

1888 de Freudenreich Isolated product from bacteria with antibacterial properties. Toxic and unstable.

Antibiotic discovery

20th Century

Erhlich Worked with dyes and arsenicals worked against Trypanosomes, very toxic.

1st antibacterial, only cured syphilis.

Domagk Research on dyes.1st synthetic antibacterial in clinical use. Prontosil cured streptococcus diseases in animals. Active component: sulphonamide group attached to dye. Toxic. Sulphonamide derivatives still used.Less toxic.

Antibiotic discovery

20th CenturyFleming and Plates left on bench over weekend.

serendipity (1928) Staphylococcus colonies lysed/killed.

Fungi beside Staphylococcus.Hypothesis: Fungi lysed

Staph.

Unable to purify in large quantities.No animal or human tests

performed.

Antibiotic discovery

20th CenturyFlorey, Chain Purified the penicillin from the fungus.

and Heatley (1939)

1940s (World War II) European and US cooperation led to increased scale production of penicillin.

Antibiotic discovery

20th centuryWaksman (1943) Isolated streptomycin from soil

bacteria Streptomyces.

Effective against Mycobacterium tuberculosis and gram negatives

.

Toxic antibiotic. Used until 1950s when isoniazid used due to shorter course of therapy.

Discovery 1928 Penicillin 1930 1932 Sulphonamides 1939 Gramicidin 1940 1942 1943 Streptomycin & Bacitracin 1945 Cephalosporins 1947 Chloramphenicol & Chlorotetracycline 1949 Neomycin 1948 Trimethoprim 1950 Oxytetracycline 1952 Erythromycin 1956 Vancomycin 1957 Kanamycin 1960 1961 Nalidixic acid 1963 Gentamicin 1964 1966 1967 Clindamycin 1968 1970 1971 Tobramycin 1972 Cephamycins & Minocycline 1980s Fluoroq uinolones 1990s Oxazolidinones

General Principles

• Selective toxicity

– The essential property of an antimicrobial drug that equips it for systemic use in treating infections is selective toxicity

– Drug must inhibit microorganism at lower concentrations than those that produce toxic effects in humans

– No antibiotic is completely safe

General Principles

• Oral and Parental

– Oral antibiotics must be able to survive stomach acid

– Advantage: Ease and reduced cost

– Disadvantage: Circuitous route, antibiotic passes to lower bowel

– Parental antibiotics given by i.v.

– Advantage: Direct route to site of infection

– Disadvantage: Increased cost and need for qualified staff

General Principles

• Half-Lives

– The length of time it takes for the activity of the drug to reduce by half

– Short half lives require frequent dosing

– Old antibiotics have short half lives

– New antibiotics may have half lives up to 33 hours

General Principles

• Broad and Narrow spectrum antimicrobials

– Broad spectrum antibiotics inhibit a wide range of bacteria

– Narrow spectrum antibiotics inhibit a narrow range of bacteria

– Broad spectrum desirable if infecting organism not yet identified

– Narrow spectrum preferable when organism has been identified

General Principles

• Bactericidal or bacteriostatic action

– Bactericidal antibiotics kill bacteria

– Bacteriostatic antibiotics inhibit the bacterial growth

– Bacteriostatic antibiotics may work as well as bactericidal antibiotics if they sufficiently arrest the bacterial growth to enable the immune system to eliminate the bacteria

General Principles

• Combinations of antibiotics

– Some antibiotics work better together than alone

– Combining 2 or more drugs may be required to prevent the emergence of resistance e.g. tuberculosis

– Combinations should not be given when 1 drug would suffice• Antagonistic effects

• No ability to adjust 1 drug concentration

Modes of action

Antimicrobial agents inhibit 5 essential bacterial processes:

1. Protein synthesis2. Folic acid synthesis3. DNA synthesis4. RNA synthesis5. Cell wall synthesis

1. Protein synthesis inhibitors

Protein synthesis

DNA mRNA Protein transcription translation

Ribosome is a protein factory in bacteria takes mRNA in and produces proteins from them.

Bacterial ribosome has 2 parts: – 30S binds to mRNA to translate mRNA into amino acids, which form

proteins– 50S required for peptide elongation

3 phases from mRNA to protein– Initiation– Elongation– Termination

Protein synthesis inhibitors

– Aminoglycosides

– Macrolides/Ketolides

– Tetracyclines

– Lincomycins

– Chloramphenicol

– Oxazolidinones

Protein synthesis inhibitors

• Bind irreversibly to ribosome

• Ribosome cannot bind to mRNA to form amino acid chains (30S) or elongate the chains to form proteins (50S)

• Disruptive effect on many essential bacterial functions leading to cell death

2. Folic acid synthesis inhibitors

pterdine + para-amino benzoic acid

dihydropterate

dihydrofolate

tetrahydrofolate

DNA/RNA

Trimethoprim

(Diaminopyrimidines)

Binding

Sulphamethoxazole

(Sulphonamides)

Structural analogues of PABA

Dihydropteroate synthetase

Dihydrofolate reductase

Reasons for combining Trimethoprim and Sulphonamides

• There is synergy between the two drugs - the combined effect is greater that the expected sum of their activities

• Individually the drugs are bacteriostatic; however, in combination they are bactericidal

• The use of two drugs will delay the emergence of resistance

3. DNA synthesis inhibitors

• Enzymes required for DNA replication

• Topoisomerase II (DNA gyrase): GyrA and GyrB

• Topoisomerase IV: ParC and ParE

• Quinolones interact/bind to the topoisomerases, which stops DNA replication e.g. nalidixic acid, ciprofloxacin

Action of fluoroquinolones

GyrA/GyrB

ParC/ParE

DNA gyrase

Quinolones

DNA

Cell death

Topoisomerase IV

DNA synthesis inhibitors

• Metronidazole

– Nitro group is reduced by bacterial enzyme

– Produces short-lived, highly cytotoxic free radicals that disrupt the DNA

– Similar effect to UV radiation on cell DNA

4. RNA synthesis inhibitors

• Rifampicin

• Forms a stable complex with bacterial DNA-dependent RNA polymerase

• Prevents chain initiation process of DNA transcription

• Mammalian RNA synthesis not affected as RNA polymerase is much less sensitive to rifampicin

5. Cell wall synthesis inhibitors

– Vancomycin

– Bacitracin

– β-lactams• Penicillins• Cephalosporins• Carbapenems• Monobactams

– β-lactamase inhibitors• Clavulanic acid• Sulbactam• Tazobactam

Action of Cell wall synthesis inhibitorsN-acetyl-glucosamine (NAG)

N-acetyl-muramic acid (NAMA)

Phospho-enol pyruvate

L-alanineD-glutamic acidL-lysine

NAMA

L-ala-D-glu-L-lys

NAMA

L-ala-D-glu-L-lys-D-ala-D-ala

D-ala-D-ala D-ala L-ala

Peptidoglycan formation

1. Building Blocks

Action of Cell wall synthesis inhibitors

Bacitracin inhibits

NAMA

L-ala-D-glu-L-lys-D-ala-D-ala

NAG

NAMA - NAG

L-ala-D-glu-L-lys-D-ala-D-ala

Lipid carrier

5 gly

NAMA - NAG

L-ala-D-glu-L-lys-D-ala-D-ala

5 gly

Phospholipid Vancomycin &Teicoplanin binds, prevents enzyme polymerisation

Action of Cell wall synthesis inhibitors

NAMA

L-ala

D-glu

L-lys

D-ala

D-ala

NAG

5 gly

NAMA

L-ala

D-glu

L-lys

D-ala

D-ala

NAG

5 gly

NAMA

L-ala

D-glu

L-lys

D-ala

D-ala

NAG

5 gly

NAMA

L-ala

D-glu

L-lys

D-ala

D-ala

NAG

5 gly

NAMA

L-ala

D-glu

L-lys

D-ala

D-ala

NAG

5 gly

Polymerisation

Action of Cell wall synthesis inhibitorsTranspeptidation

NAMA

L-ala

D-glu

L-lys

D-ala

D-ala

NAG

5 gly

NAMA

L-ala

D-glu

L-lys

D-ala

D-ala

NAG

5 gly

NAMA

L-ala

D-glu

L-lys

D-ala

D-ala

NAG

5 gly

NAMA

L-ala

D-glu

L-lys

D-ala

D-ala

NAGNAMA

L-ala

D-glu

L-lys

D-ala

D-ala

NAGNAMA

L-ala

D-glu

L-lys

D-ala

D-ala

NAG

-lactams resemble D-ala-D-ala, bind to enzyme, inhibit cross-linking

Penicillin Binding ProteinsEnzymes involved in cell wall formation

• Reseal cell as new peptidoglycan layers added

• Penicillins bind to PBPs block enzyme cross-linking chains

• Weak cell wall

• Build up osmotic pressure

• Lysis

Keynote points

• Recent history of antibiotic discovery

• General principles of antibiotic action

• 5 modes of action

• Examples of each