Lecture 19B - Antibiotics B

Question 1

What is tetrahydrofolate (THF)?

Answer 1

Cofactor essential for synthesis of
precursors of DNA, RNA, protein,
fatty acids and vitamins in bacteria
and humans, and for
N-formylmethionine (fMet) in
bacteria (initiation of protein
synthesis)

Question 2

Precursor for THF?

Answer 2

dihydrofolic acid (DHF)

Question 3

Precursor for DHF?

Answer 3

p-aminebenzoic acid (PABA)

Question 4

Enzymes that convert PABA to DHF?

Answer 4

Pteridine synthetase and DHF synthetase

Question 5

What is the difference in how bacteria synthesize DHF compared to mammals?

Answer 5

Mammalian cells use Folic Acid (Folate) obtained from food while bacteria synthesize it from PABA -> dihydropteroic acid -> DHF

Question 6

What do sulfonamides target?

Answer 6

Structurally similar to
PABA – they are competitive
inhibitors of pteridine synthase in
bacteria

Question 7

What do trimethoprim target?

Answer 7

Structurally similar
to DHF – it acts as a competitive
inhibitor of the microbial DHF
reductase but not the mammalian
enzyme

Question 8

How does resistance develop against sulfonamides?

Answer 8

Mutation in pteridine synthetase such that it no longer binds but is still functional (immunity), some bacteria can make THF directly from folic acid like mammals (bypass)

Question 9

How does resistance develop against trimethoprim?

Answer 9

Mutation in DHF reductase such that it no longer binds but is still functional (immunity), acquisition of a plasmid-encoded DHF reductase that doesn’t bind trimethoprim (bypass)

Question 10

How does metronidazole work?

Answer 10

damages DNA, inhibits DNA

synthesis

Question 11

How is metronidazole activated?

Answer 11

By bacterial
housekeeping proteins flavodoxin
and ferredoxin in microaerophilic
and obligate anaerobes

Question 12

Why is metronidazole only activated in bacterial cells?

Answer 12

Lack flavodoxin and ferredoxin

Question 13

Which bacteria is metronidazole used for?

Answer 13

Helicobacter pylori and Clostridium difficile, also effective against some eukaryotic pathogens that can activate metronidazole

Question 14

Which bacteria are sulfonamides and trimethoprim used for?

Answer 14

Many species - broad spectrum

Question 15

How does resistance develop against metronidazole?

Answer 15

Upregulation of DNA repair enzymes (RecA) (bypass/immunity)

Question 16

How do quinolones and fluoroquinolones work?

Answer 16

Inhibit DNA replication, quinolones bind to and stabilize the gyrase:DNA complex in DNA gyrase and prevent strand resealing, resulting in double strand breaks in the DNA.

Question 17

What do quinolones/fluoroquinolones target?

Answer 17

DNA gyrase (Topoisomerase II)

Question 18

Difference between quinolones and fluoroquinolones?

Answer 18

Fluoroquinolones are just fluorinated, more active and effective quinolones

Question 19

Which bacteria is ciproflaxin(quinolone) used for?

Answer 19

B anthracis (anthrax)

Question 20

How is resistance developed against quinolones/fluoroquinolones?

Answer 20

Single mutation in DNA gyrase can make bacteria resistant - gyrase still active but no longer binds (immunity)

Question 21

What does rifampicin target?

Answer 21

Exit channel on RNA polymerase beta subunit

Question 22

How does rifampicin work?

Answer 22

Blocks emergence of new RNA from exit channel in bacterial RNA pol beta subunit

Question 23

How is resistance developed against rifampicin?

Answer 23

Single mutation in
RNA polymerase that prevents rifampin binding
but not RNA polymerization.

Question 24

What do aminoglycosides like streptomycin target?

Answer 24

Bind to the bacterial ribosomal 30S subunit, inhibit the initiation and
elongation steps in protein synthesis. Allows the wrong tRNA to bind inhibiting protein synthesis.

Question 25

How does resistance develop against aminoglycosides?

Answer 25

Aminoglycosidal modifying enzymes - modification reduces their transport into cell (too big) and their ability to bind to the 30S subunit

Question 26

What do tetracycline target?

Answer 26

Bind to the 30S subunit and inhibits tRNA binding at the A site

Question 27

How does tetracycline work?

Answer 27

Distort A site and prevent alignment of aminoacylated tRNA with codon on mRNA – block peptide synthesis

Question 28

How is resistance developed against tetracycline?

Answer 28

Efflux pumps, modification by tetracycline enzymes, cytoplasmic proteins that protect 30S subunit by preventing binding without disrupting protein synthesis

Question 29

What do chloramphenicol target?

Answer 29

Binds to the peptidyl transferase on the 50S subunit, inhibits the peptidyl transfer step in protein synthesis

Question 30

How is resistance to chloramphenicol developed?

Answer 30

Bacteria have/acquire an enzyme, chloramphenicol acetyltransferase, which acetylates chloramphenicol, inactivating it.

Question 31

What do macrolides target?

Answer 31

Bind to the 50S subunit and inhibit | translocation

Question 32

What is azithromycin? What is it used for?

Answer 32

Macrolide. Very effective at treating sexually transmitted infections such as Chlamydia and N. gonorrhoeae – single dose (cf. a full course) but very expensive

Question 33

How is resistance against macrolides developed?

Answer 33

Efflux pump

Question 34

What is the difference between lincosamides/streptogramins and macrolides?

Answer 34

Differ in structure from macrolides but have the same mechanism - bind to roughly the same site on the 50S ribosome and inhibit translocation step. Lincosamides kill intestinal microbiota, associated with C difficile infections. Streptogramins used in agriculture - many resistance strains.

Question 35

Which antibiotics are used against 30S subunit of bacterial ribosome? 50S subunit?

Answer 35

30S - Tetracycline, aminoglycosides | 50S - Chloramphenicol, macrolides, streptogramins, lincosamides

Question 36

What does teixobactin target?

Answer 36

Binds to precursors of peptidoglycan – Lipid II and Lipid II precursors and prevents PBP/transpeptidase activity, blocking peptidoglycan synthesis

Question 37

Which bacteria is teixobactin effective against?

Answer 37

Gram-positive bacteria – S. aureus, S. pneumoniae, M. tuberculosis, vancomycin-resistant enterococci (VRE, have D-Ala-D-Lac but can still bind teixobactin). Gram-negatives are resistant

Question 38

How are gram-negatives resistant to teixobactin?

Answer 38

Act on PG and WTA in gram-positive bacteria which lack OM - presence of the outer membrane prevents uptake of teixobactin

Question 39

Why is resistance less likely to occur with teixobactin?

Answer 39

Acts on lipid II, a non-protein molecule - can't change lipid II

Question 40

What kinds of organisms can be found in the microbiota?

Answer 40

Bacteria, archaea, fungi, some protozoa and viruses/phages

Question 41

Why are most microbes found in the gut?

Answer 41

Provides stable nutrients and temperature

Question 42

Difference between microbiota and microbiome?

Answer 42

Microbiota - refers to the community microbes | Microbiome - refers to the combined genomes

Question 43

How are the 60-90% non-culturable gut microbiota identified?

Answer 43

(1) targeted sequencing of phylogenetically informative genes, usually 16s rRNA, a taxonomic marker for bacteria, or (2) by random sequencing of all genes in the microbial community (shotgun metagenomic sequencing of stool samples). Successful identification of bacterial species in a microbiome requires a match with existing genome sequences in databases

Question 44

Which bacteria dominate in the gut?

Answer 44

In adults, Gram-positive Firmicutes (eg. Clostridia, Lactobacilli, Entercocci) and Gramnegative Bacteroidetes dominate. Most gut bacteria are anaerobic. Some are adherent, most are in the lumen of the colon (large intestine).

Question 45

What are the metabolic functions of gut microbiota?

Answer 45

Aid in digestion by fermenting/breakdown of complex carbohydrates, amino acids, bile salts, lipids, fatty acids, salvage urea, synthesize essential amino acids, vitamins (eg. vitamin K, folic acid) – these nutrients are used by the bacteria but are also available to host, modulate appetite, etc

Question 46

What are the trophic functions of gut microbiota?

Answer 46

Facilitate gut development by controlling epithelial cell proliferation and differentiation; influence organ development, development of lymphatic tissues, angiogenesis, fat storage

Question 47

What are the protective functions of gut microbiota?

Answer 47

Protective: out-compete pathogenic bacteria for nutrients, prevent colonization by pathogens, stimulate the immune system, facilitate its maturation to eliminate pathogens but tolerate commensals

Question 48

How do C difficile infections occur?

Answer 48

May be present in the gut in low levels prior to antibiotic treatment, but controlled by competition with commensal bacteria, or may be acquired by ingestion of spores in hospital. C. difficile spores are robust and difficult to kill – are prevalent in hospital settings.

Question 49

What is C difficile?

Answer 49

Anaerobic spore-forming Gram-positive bacillus. Causes severe diarrhea, pseudomembranous colitis, toxic megacolon - can be fatal. Resistant to multiple antibiotics. Vegetative cells produce toxins TcdA, TcdB (for Toxin C. difficile A, B) that cause epithelial damage. Levels of glucose and other nutrients affect expression of C. difficile genes. Intestinal components such as bile acids induce C. difficile spores to germinate.