Lecture 18: Antimicrobials and resistance I

Question 1

What are antimicrobials and what is their general mode of action?

Answer 1

• Any substance of natural, semisynthetic or synthetic origin that kills or inhibits the growth of microorgansims such as viruses, fungi and protozoa.
• Generally small molecules that are able to diffuse into the microorganism throught the membrane via porins.

Question 2

What are antibiotics?

Answer 2

• Antibiotics are a type of antimicrobial that are generally used against bacteria.
• Antibiotics are produced by several groups of microbes (bacteria, fungi) as their natural defense system against other microbes living in their vicinity.

Question 3

What is selective toxicity?

Answer 3

• The antimicrobial molecule must be toxic to the microorganism but not the host (e.g. humans).

Question 4

What is meant by chemotherapeutic index?

Answer 4

• Chemotherapeutic dose= Toxic dose/Therapeutic dose
• Toxic dose of a drug - the concentration causing harm to the host.
• Therapeutic dose of a drug - the concentration eliminating pathogens in the host.
• Ideally, we want Therapeutic does >>> toxic dose.

Question 5

How are antimicrobials selective?

Give some examples of structures that are unique to microorganisms.

Give some examples of structures that are present in microorganims and the eukaryotic host but different enough to be targeted.

Answer 5

• Antimicrobials must target structures that are either not present or very different between the microorgansim and eukaryotic host.
• Examples include: peptidoglycan (bacteria), revrese transcriptase (retroviruses) and ergosterol (fungi).
• Proteins such as RNA polymerse, ribosomes and ribosomal stuctures

Question 6

How are antibiotics classified? (4 methods of classificaction)

Answer 6

1. Natural vs semi-synthetic vs synthetic

2. Spectrum of activity

3. Bactericidal vs bacteriostatic

4. Mode of action/specific target molecule

Question 7

What is the spectrum of activity of antibiotics?

Answer 7

• Antibiotics are classified as either broad-spectrum, intermediate-spectrum or narrow spectrum.
• Broad-spectrum antibiotics- active against both gram-positive and gram-negative bacteria.
• Narrow-spectrum antibiotics- limited activity and are primarily only useful against specific microorganisms.
• The spectra of activity may change as various bacteria aquire resistance to these antibiotics.

Question 8

What is the difference betwwen bactericidal and bacteriostatic?

Answer 8

• Bactericidal antibiotics kill the bacteria.
• Bacteriostatic antibiotics inhibit bacterial growth (growth arrest). Thia allows the immune system to overcome the non-growing bacteria.

Question 9

What is peptidoglycan?

Answer 9

• An essential molecule in the cell wall of gram-positive (thick peptidoglycan layer) and gram negative bacteria (thin peptidoglycan layer).
• It gives structural rigidity to the cell.

Question 10

Describe the structure of peptidoglycan.

Answer 10

• Backbone structure: N-acetylglucosamine (NAG) linked β-1,4 to N-acetylmuramic acid (NAM)
• Each NAM has an attached chain of 4-5 amino acids; cross-linking between these amino acids gives peptidoglycan its strong structure.
• Prior to cross-linking, each side chains ends in D-Ala-D-Ala.

Question 11

How do antibiotics target cell wall synthesis?

Answer 11

• Target peptidoglycan- either by binding to and inactivating the transpeptidase enzymes that are essential for cross-linking peptidoglycan (e.g. penicillin) or by directly binding to peptidoglycan units and stopping protein (enzyme) binding and therefore cross-linking (e.g. vancomycin).
• These antibiotics are bactericidal against growing cells.

Question 12

How do β-lactams (e.g penicillin) inhibit crosslinking of peptidoglycans?

Answer 12

• β-lactams mimic the D-Ala-D-Ala structure.
• E.g. penicillin- suicide inhibitor of the transpeptidase enzyme; due to the similar structure, penicillin can bind to and inactivate the transpeptidase enzyme so that it does not bind to the D-Ala-D-Ala links on the peptidoglycan and therefore cannot form cross-links.
• β-lactams form covalent bonds with the OH group on serine in the active site of the transpeptidase enzyme.
• Different classes of the β-lactams share the core structure enabling them to bind to and inhibit transpeptidase activity.

Question 13

How does vancomycin target cell wall synthesis of bacteria?

Answer 13

Vancomycin binds directly to the D-Ala-D-Ala components of peptidoglycan, therefore, the transpeptidase enzymes cannot access these parts and this leads to lack of cross-link formation between peptidoglycan units.

Question 14

What are the three types antibiotics that work by inhibiting protein synthesis? Give examples of each type.

Answer 14

1. Macrolides:
   - e.g. erythromycin, clarithromycin
2. Tetracyclines
   - e.g. tetracycline, doxycycline
3. Aminoglycosides
   - e.g. Sreptomycin neomycin

Question 15

How do antibiotics target protein synthesis? Give examples.

Answer 15

• Linezolid- binds to 50S of ribosome and prevents formation of the 50S/30S ribosomal complex.
• Tetracyclines- bind to 30S of ribosome and interfere with binding of tRNA to ribosomal subunit.
• Chloramphenicol- binds to 50S of ribosome and prevents formation of peptide bond between amino acids.
• Aminoglycosides- bind to 30S of ribosome and cause mRNA codon to be misread; interfere with initiation complex fo 30S and 50S with mRNA.
• Macrolides and streptogramins- block the polypeptide exit tunnel on th 50S of ribosome (5^’ end) and prevents peptide chain elongation.

Question 16

Which antibiotics target the cytoplasmic membrane of bacteria?

Answer 16

• Cyclic lipopeptides such as polymyxin and daptomycin.
• Polymyxin is more positively charged than daptomycin.

Question 17

How does polymyxin target bacterial membranes?

Answer 17

• Only active in gram negative bacteria as it interacts with the negatively charged lipopolysaccharide (LPS) and is then inserted into the outer membrane.
• The positively charged region of polymyxin interacts with negatively charge phosphate group on LPS.
• Polymyxin is embedded into outer membrane, this causes exclusion of ions such as Mg²⁺ and Ca²⁺ which stabilize the LPS; this leads to destabilization of outer membrane.
• The lipid region/ hydrophobic region of polymyxin alloews it to move through the membrane and pass through the inner membrane.
• Overall, this leads to complete distruption of outer membrane, cell leakage and eventually cell death.

Question 18

How does daptomycin target bacterial cytoplasmic membranes?

Answer 18

• Daptomycin mainly targets gram positive bacteria as it does not interact with the LPS the same wa as polymyxin.
• Daptomycin has specifity for the phospholipids in gram positve bacteria.
• It integrates into the cytoplasmic membrane and distrupts membrane structure, causes leakage and may also misregulate localisation of cell division proteins.

Question 19

1. Give examples of antibiotics that target bacteria via targeting DNA-dependant RNA polymerase.
2. How do these antibbiotics target DNA-dependant RNA polymerase?
3. Are these antibiotics bacteriostatic or bactericidal?
4. What are some properties of these antibiotics?

Answer 19

1. Rifampicin/rifampin, rifabutin, rifapentine.
2. Bind directly to DNA-dependant RNA polymerase and block mRNA synthesis.
3. These antibiotics are bactericidal and generally broad spectrum
4. Well absorbed orally, can cross into cells (effective for intracellular bacteria e.g. mycobacterium infections), can cross blood-brain barrier.

Question 20

Why is it hard to find anti-fungal agents that are fully specific to the fungi?

Answer 20

• Fungi are eukaryotic cells.
• Many of the proteins in fungi are structurally similar to proteins found in host eukaryotic cells.

Question 21

What are some examples of antifungal agents and what is their mechanism of action?

Answer 21

• Echinocandins- inhibition of glucan cell wall synthesis.
• Griseofulvine- Inhibition of microtubule synthesis in a way that is specific to fungal cells.
• Flucytosine- Inhbition of nucleic acid synthesis.

Question 22

How is ergosterol tageted by antifungal agents?

Answer 22

• Ergosterol-sterol found in cell membranes of fungi and protozoa, has a similar function to cholesterol in animal cells.
• Essential in most fungi.
• Azoles and terbinafine target ergosterol synthesis by targeting molecules in the synthesis pathway
• Amphotericin B and nystatin target membrane permeability by directly binding to ergosterol and increasing membrane permeability and causing cell death.