Anti-microbial chemotherapy - agents and mechanisms of action

Question 1

Antimicrobial agents

Answer 1

Aimed at controlling specific infecting organisms
-narrow spectrum
-broad spectrum
‘Selective toxicity’

Question 2

Antimicrobial agents are therapeutically useful if the target

Answer 2

Is not present in man
If microorganisms has higher affinity for the drug than man
Most antibiotics in clinical usage directed against bacterial cell wall synthesis, bacterial protein synthesis, or bacterial nucleic acis synthesis, which are unique in some ways to bacteria

Question 3

Selective toxicity

Answer 3

Must be highly effective against microbe but have minimal or no toxicity to humans.
In practice, this is expressed by a drug’s therapeutic index (TI) - the ratio of the toxic dose (to the patient) to the therapeutic dose (to eliminate the infection).
The larger the index, the safer is the drug (antibiotic) for human use.

Question 4

A clinically-useful antibiotic should have as many of these characteristics as possible:

Answer 4

It should have a wide spectrum of activity with the ability to destroy or inhibit many different species of pathogenic organisms.
It should be nontoxic to the host and without undesirable side effects.
It should be nonallergenic to the host.
It should not eliminate the normal flora of the host.
It should be able to reach the part of the human body where the infection is occurring.
It should be inexpensive and easy to produce.
It should be chemically-stable (have a long shelf-life).
Microbial resistance is uncommon and unlikely to develop

Question 5

Therapeutic index

Answer 5

Bigger the number, the safer the drug

The ratio of the toxic dose (to the patient) to the therapeutic dose (to eliminate the infection)

Question 6

Classification of antimicrobials

Answer 6

By chemical structure
-e.g. b-lactam ring (e.g. penicillin)
By target site
According to whether they are bactericidal (kill) or bacteriostatic (inhibit growth)
-distinction is blurred

Question 7

Testing antibiotics

Answer 7

Disc diffusion on agar
-lawn of bacteria
-filter paper disc placed
-antibiotic diffuses out, can see if bacteria is killed or not
In liquid - MIC/MBC testing

Question 8

MIC/ MBC

Answer 8

Minimal inhibitory concentration
Minimal bactericidal concentration
-put certain amount of antibiotic in, then leave overnight
-see if any growth/ colonies

Question 9

Main targets for antimicrobials

Answer 9

Cell wall - peptidoglycan (most effective against gram positive)
Protein synthesis - ribosomes or enzymes
Metabolic pathways
DNA
Membranes
Enzymes

Question 10

Cell wall

Answer 10

Peptidoglycan is a unique structure (humans don’t have it)

• needs to be a cross-linked structure
• amino acid crosslinks e.g. d-ala, d-glutamate - a lot of antimicrobials act against crosslinking

Question 11

Main classes of agent that act against cell wall

Answer 11

Main classes of agent are

• b-lactams – penicillins and cephalosporins
• glycopeptides – vancomycin, teicoplanin
• cycloserine – inhibits alanine racemase & D-alanine ligase

Question 12

b-lactam antibiotics

Answer 12

Beta-lactams - bactericidal compounds
Contain a b-lactam ring and inhibit normal cell wall formation by inhibiting PBPs that cross-link amino acids to form peptidoglycan wall
Beta-lactam ring- can have different structures attached
-1) Penicillins - five-membered
-2) Cephalosporins - six-membered

Question 13

Structure of penicillin (action)

Answer 13

Mimics structure of D-ala-D-ala
Inhibits formation of peptidoglycan cross-links in bacterial cell wall by binding of 4-membered β-lactam ring of penicillin to the enzyme DD-transpeptidase (penicillin binding protein (PBP))
DD-transpeptidase cannot then catalyze formation of these cross-links&raquo_space;> 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

Vancomycin resistant bacteria

Answer 15

Staph aureus resistance becoming more common

Question 16

Antiobiotics interfere with protein synthesis

Answer 16

Tetracyclines inhibit tRNA binding
Aminoglycosides bind to 30s subunit and cause misreading of genetic code
Erythromycin binds to molecules in 50s subunit blocking exit of nascent polypeptide chain
Fusidic acid binds to EFG (bacterial protein needed for translocation on becaterial ribosome after peptide bond formation) therefore prevents protein synthesis

Question 17

Aminoglycosides

Answer 17

Aminoglycosides – contain an aminocyclitol ring linked to a sugar
-e.g. gentamicin, streptomycin
Effective against aerobes and facultative anaerobes
-not active against anaerobes
Not effective against anaerobes as bacterial up-take requires oxygen- or nitrate-dependent electron transport
Not absorbed from the gut
-must be given intravenously or intramuscularly for systemic treatment
-side effects – nephrotoxicity, ototoxicity

Question 18

Facultative vs strict anaerobes

Answer 18

Facultative - oxygen dependent or independent pathway

Strict- die in presence of oxygen

Question 19

Tetracyclines

Answer 19

Bacteriostatic compounds
-all broad spectrum
-penetrates mammalian cells to reach intracellular organisms
-incorporated into developing bone and teeth
Use restricted due to widespread resistance

Question 20

Macrolides

Answer 20

Bind to 50S subunit blocking exit of nascent polypeptide chain
A family of large cyclic molecules all containing a macrocyclic lactone ring
-bacteriostatic
-erythromycin most commonly used
Erythromycin used for penicillin allergic patients
-penetrates mammalian cells to reach intracellular organisms

Question 21

Agents affecting DNA

Answer 21

Quinolones - target site is DNA gyrase
Rifamycins - inhibit DNA dependent RNA synthesis due to high affinity for bacterial RNA polymerase (poor affinity for mammalian)
Metronidazole - disrupts DNA - anaerobes - direct damage to DNA e.g. strand breakage

Question 22

Nitroimidazoles

Answer 22

Disrupt DNA
Metronidazole, Tinidazole - antiparasitic and antibacterial properties
-originally introduced for the treatment of the flagellate parasite Trichomonas vaginalis

Question 23

Metronidazole is inactive

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

Question 24

Metronidazole is active only against strictly anaerobic organisms

Answer 24

because only these can produce the low redox potential necessary to reduce the drug

Question 25

Metabolic pathways - folic acid synthesis

Answer 25

Folic acid enzymes – needed for amino acid (& so protein) synthesis
Some antibiotics, e.g sulfonamides such as sulfanilamide, interrupt these enzymes
Active against Gram + and Gram –
Structural analogues - act in competition
Resistance becoming a problem

Question 26

Antibiotic resistance definition

Answer 26

an 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

Antibiotic resistance types

Answer 27

Some resistance is by chromosomal mutation, some is coded for by plasmid DNA
Some plasmids are transmissible
Transposons can carry resistance genes and jump between chromosome and plasmid

Question 28

Transfer of resistance

Answer 28

‘Cassettes’ of multiple resistance genes are sometimes organized into genetic elements called integrons
The integron contains a gene for a recombination enzyme to allow insertion (and excision)
-mutant selection
-plasmid mediated resistance
-plasmid mediated on transposon

Question 29

Antimicrobial resistance - different ways

Answer 29

Target is structurally altered (by mutation)
-now has lower affinity for antibacterial
-pencillin-binding proteins
Target overproduced
-dihydropteroate synthetase (bacterial target) and sulphonamide
Drug not activated
-aerobes and metronidazole
Drug is removed
-enzymic destruction
(b-lactamase; 3 modifying enzymes responsible for acquired aminoglycoside resistance)
-efflux (tetracyclines, quinolones)
Drug cannot gain entry to cell
outer-membrane barrier, lack of transport mechanism

Question 30

Antivirals

Answer 30

Few in number & narrow in spectrum.
Virustatic not virucidal
Need to interfere with viral machinery without affecting host

Question 31

Methods of antivirals

Answer 31

1) Penetration/uncoating
2) Taking over cell machinery
3) Post-translation inhibition

Question 32

Antivirals - post-translation inhibition

Answer 32

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

Question 33

Antivirals - taking over cell machinery

Answer 33

-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 34

Antivirals - penetration/ uncoating

Answer 34

• amantadine

- prevent fusion of viral envelope with cell membrane