Antimicrobials 1: Origins and modes of action

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Antimicrobials 1 Origins and modes of action

• Dr Fiona Walsh

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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

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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

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Antibiotic discovery

• 19th Century
• Louis 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.

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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.

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Antibiotic discovery

• 20th Century
• Fleming 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.

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Antibiotic discovery

• 20th Century
• Florey, 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.

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Antibiotic discovery

• 20th century
• Waksman (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.

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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

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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

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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

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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

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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

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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

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Modes of action

• Antimicrobial agents inhibit 5 essential
• bacterial processes
• Protein synthesis
• Folic acid synthesis
• DNA synthesis
• RNA synthesis
• Cell wall synthesis

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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

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Protein synthesis inhibitors

• Aminoglycosides
• Macrolides/Ketolides
• Tetracyclines
• Lincomycins
• Chloramphenicol
• Oxazolidinones

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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

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2. Folic acid synthesis inhibitors

• pterdine para-amino benzoic acid
• dihydropterate
• dihydrofolate
• tetrahydrofolate
• DNA/RNA

Sulphamethoxazole (Sulphonamides) Structural
analogues of PABA
Dihydropteroate synthetase
Dihydrofolate reductase
Trimethoprim (Diaminopyrimidines) Binding
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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

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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

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Action of fluoroquinolones
GyrA/GyrB
DNA gyrase
DNA
ParC/ParE
Topoisomerase IV
Quinolones
Cell death
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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

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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

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5. Cell wall synthesis inhibitors

• Vancomycin
• Bacitracin
• ß-lactams
• Penicillins
• Cephalosporins
• Carbapenems
• Monobactams
• ß-lactamase inhibitors
• Clavulanic acid
• Sulbactam
• Tazobactam

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Action of Cell wall synthesis inhibitors
N-acetyl-glucosamine (NAG)
Phospho-enol pyruvate
Peptidoglycan formation 1. Building Blocks
N-acetyl-muramic acid (NAMA)
L-alanine D-glutamic acid L-lysine
D-ala-D-ala
D-ala
L-ala
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Action of Cell wall synthesis inhibitors
Lipid carrier
NAG
Bacitracin inhibits
5 gly
Vancomycin Teicoplanin binds, prevents enzyme
polymerisation
Phospholipid
5 gly
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Action of Cell wall synthesis inhibitors
Polymerisation
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Action of Cell wall synthesis inhibitors
Transpeptidation
b-lactams resemble D-ala-D-ala, bind to enzyme,
inhibit cross-linking
D-ala
D-ala
D-ala
D-ala
D-ala
D-ala
L-lys
L-lys
L-lys
D-glu
D-glu
D-glu
L-ala
L-ala
L-ala
NAMA
NAG
NAMA
NAG
NAMA
NAG
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Penicillin Binding Proteins Enzymes 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

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Keynote points

• Recent history of antibiotic discovery
• General principles of antibiotic action
• 5 modes of action
• Examples of each