Antimicrobial chemotherapy - agents and mechanisms of action

Question 1

When are antimicrobial agents therapeutically useful?

Answer 1

Useful if the target is;

• not present in man
• if microorganism has higher affinity for the drug than man
• most antibiotics in clinical usage are directed against bacterial cell wall synthesis, bacterial ps or bac nucleic acid synthesis

Question 2

Selective toxicity?

Answer 2

• Must be highly effective against microbe but have minimal or no toxicity to humans
• In practice it is expressed y a drug’s therapeutic index (TI) - ratio of the toxic dose to the pt to the therapeutic dos to eliminate the infec
• larger the index = safer the drug is

Question 3

What are antimicrobial agents? Types?

Answer 3

Can be narrow (either gram pos or neg) or broad spectrum (against gram pos and neg)
Control specific infecting organisms

Question 4

Desirable characteristics of antibiotics?

Answer 4

• Wide spectrum activity and can destroy or inhibit many different species of pathogenic organisms
• Non-toxic to host
• No undesirable side effects
• Non-allergenic
• Reach body part with infec
• Not eliminate normal flora of host
• Cheap, easy to produce

Question 5

How are antimicrobials classified?

Answer 5

By chemical structure, target site and according t whether they are bactericidal (kill) or bacteriostatic (inhibit growth)

Question 6

What are the main targets for antimicrobials?

Answer 6

• Cell wall - peptidoglycan
• PS - ribosomes or enzymes
• Metabolic pathways
• DNA
• Membranes
• Enzymes

Question 7

How to test for antibiotics?

Answer 7

Disc diffusion on agar - if bac sensitive to antibiotic = clear zones formed

Question 8

How to test for antibiotics in liquid?

Answer 8

MIC/MBC test
MIC = minimal inhibitory conc = min antibiotic needed to inhibit bac growth = conc that stops growth = clear broth
MBC - minimal bactericidal conc = min. to kill bac = lowest conc where no colonies formed = clear plate
Add same amount of bac to each tube and decrease conc of antibiotic in each tube

Question 9

Cell wall targets?

Answer 9

Peptidoglycan = unique, needs to be crosslinked = AA crosslinked

Question 10

What are the main classes of agents that act against the cell wall?

Answer 10

• Beta-lactams - penicillins and cephalosporins
• Glycopeptides - vancomycin
• Cycloserine - inhibits alanine racemase

Question 11

Beta-lactam antibiotics?

Answer 11

Bactericidal compounds, contain beta-lactam ring and inhibit normal cell wall formation
Beta-lactam ring can have diff structures attached;
- penicillins - 5 membered
- cephalosporins - six-membered

Question 12

What do beta-lactam’s do?

Answer 12

Inhibit peptidoglycan formation

Question 13

How does penicillin’s structure aid it’s function?

Answer 13

Mimics structure of D-ala-D-ala = inhibits formation of peptidoglycan cross-links in the bacterial cell wall by binding of the 4-membered beta-lactam ring of penicillin to the enzyme DD-transpeptidase
DD-transpeptidase cannot then catalyse formation of these crosslinks = cell death

Question 14

Vancomycin?

Answer 14

Effective against Gram positive organisms
Binds to D-alanyl D-alanine dipeptide on side chain of newly synthesised peptidoglycan subunits, preventing them from being incorporated into cell wall by penicillin binding proteins (PBPs)

Question 15

How do antibiotics inhibit protein synthesis?

Answer 15

• Aminoglycosides – bind to 30S subunit & cause misreading of genetic code
• Tetracyclines – inhibit binding of tRNA to mRNA – ribosome complex
• Erythromycin – binds to a molecule in 50S subunit blocking exit of nascent polypeptide chain
• Fusidic acid – ‘elongation factor G (EFG)’ – bacterial protein needed for translocation on bacterial ribosome after peptide bond formation during protein synthesis. Fucidic acid binds EFG preventing protein synthesis.

Question 16

Aminoglycosides?

Answer 16

Bind to 30S subunit = misreading of genetic code
contain an aminocyclitol ring linked to a sugar
Effective against aerobes and facultative anaerobes
Not active against anaerobes

Question 17

Why are aminogylcosides not effective against anaerobes?

Answer 17

Bacterial up-take requires oxygen- or nitrate-dependent electron transport

Question 18

Problem with aminoglycosides?

Answer 18

Not absorbed from the gut
must be given intravenously or intramuscularly for systemic treatment
Side effects – nephrotoxicity, ototoxicity

Question 19

Tetraclyclines?

Answer 19

Inhibit binding of tRNA to mRNA/ribosome complex

• Bacteriostatic compounds = all broad spectrum, penetrate mammalian cells = reach intracellular organisms, incorporated in developing bone and teeth
• Use restricted due to resitance

Question 20

Macrolides?

Answer 20

• Bind to 50S subunit blocking exit of nascent polypetide chain
• Large cyclic molecules containing macrocyclic lactone ring - bacteriostatic
• Erythromycin most commonly used = e.g. for penicillin allergic pts - penetrates mammalian cells to reach intracellular organisms

Question 21

What are the agents that affect DNA? How?

Answer 21

• Quinolones - Target site is DNA gyrase – DNA gyrase is a topoisomerase that relieves strain while DS DNA is being unwound by helicase.
• Rifamycins – inhibit DNA dependent RNA synthesis due to high affinity for bacterial RNA polymerase (poor affinity for mammalian)
• Metronidazole - disrupts DNA – only works when it is reduced and as this reaction only usually happens in anaerobic cells – effective against anaerobes

Question 22

Nitroimidazoles?

Answer 22

Disrupt DNA

E.g metronidazole, tinadazole = antiparasitic and antibac properties

Question 23

How does metronidazole work?

Answer 23

• Activated in cell by redox enzyme pyruvate-ferredoxin oxidoreductase
• In anaerobes, ferredoxin is an e- transporter molecule that reduces (gives electrons to) Metronidazole
• This single electron transfer reduces nitro group of met. creating highly reactive anion – disrupts DNA helix
  intermediate is short-lived and decomposes
• Metronidazole is active only against strictly anaerobic organisms
  because only these can produce the low redox potential necessary to reduce the drug.

Question 24

Which antibiotics interfere with metabolic pathways?

Answer 24

• Folic acid enzymes = needed for AA synthesis and PS
• E.g Sulfonamides = inhibit these enzymes
• Active against gram pos and neg

Question 25

Problem with metabolic pathway antibiotics?

Answer 25

Resistance

Question 26

Define antibiotic resistance

Answer 26

Organism that is not inhibited or killed by an antibacterial agent at concentrations of the drug achievable in the body after normal dosage.

Question 27

How is antibiotic resistance developed?

Answer 27

Chromosomal mutation
Coded for by plasmid DNA
Plasmids are transmissible
Transposons can carry resistance genes and jump between chromosome and plasmid

Question 28

How is antibiotic resistance transferred?

Answer 28

Cassettes of multiple resistance genes can be organised into genetic elements called integrons
Integrons contain gene for recomination enzyme to allow insertion

Question 29

Mechanisms of antibiotic resistance?

Answer 29

• The target is structurally altered (by mutation)
  Now has a lower affinity for the antibacterial
  Pencillin-binding proteins

- The target is overproduced
Dihydropteroate synthetase (bacterial target) and sulphonamide

• The drug is not activated
  Aerobes and metronidazole
• The drug is removed
  Enzymic destruction
  β-lactamase
  3 modifying enzymes responsible for acquired aminoglycoside resistance
• Efflux
  Tetracyclines, quinolones
• The drug cannot gain entry to the cell
  outer-membrane barrier, lack of transport mechanism

Question 30

Antivirals?

Answer 30

Few in number
Narrow spectrum
Virustatic
Need to interfere with viral machinery without effecting host

Question 31

How do antivirals work?

Answer 31

1)Penetration/uncoating
Amantadine
Prevent fusion of viral envelope with cell membrane
2) Taking over cell machinery
- Transcription
Nucleoside analogues - Zidovudin, acyclovir
Zidovudin (AZT) acts as substrate for & inhibitors of viral reverse transcriptase
Acyclovir – inhibits HSV DNA polymerase
- Translation
anti-sense morpholinos (oligomer molecules that block target sequence in RNA by binding); ribozymes (cut viral RNA)

Question 32

Post-translation inhibition of antivirals?

Answer 32

Post-translation inhibition;

• Protease inhibitors
• Protease cleaves viral polyproteins into structural proteins required for viral replication
• Protease inhibition – immature, defective viral particles - HAART (highly active antiretroviral therapy)