Lec 12-Anti-microbial Resistance

Question 1

Antibiotics are natural products

Answer 1

• NP often have many saturated carbons & are thus more three dimensional rather than flat
• Np- often have many functional groups (H-bond donors and acceptors etc) and are intrinsically more likely to interact with proteins
• Often have complex fused ring system or macrocyclic rings
• Often have many chiral centres
• SIZE= higher MW typically
• Typically synthetically complex and demanding, often needing years to develop an effective synthesis

Answer 2

Question 4

Mechanisms underlying resistance

Answer 4

• Many different mechanisms for resistance
  *

Question 5

Microbes evolve quickly

Answer 5

1. Generation time of bacteria is very short
   
   • E.Coli = 17 minutes; Staph.Aureus = 30 min; TB= 850 min; Treponema Pallidum = 1980 min
   • q.v. 1 year or 20 years for large animals
   • E.coli evole 620,000 times faster than us
2. Horizontal transfer of proteins or whole collections of proteins
   
   • Millions of years of evolution in a moment

Question 6

Multi-resistant organism

Answer 6

• Pseudomonas Aeruginosa
• Burkholderia Cepacia
• Stenotrophomonas Maltophilia
• Acinetobacter Baumanii
• Mycobacterium Tuberculosis
• Mycobacterium Avium-intracellulare

Question 7

Bacterial resistance to antimicrobial drugs is an increasing health and economic problem

Answer 7

• Innate resistance- To survive war microbes must be robust in the face of small molecules
• Acquired resistance- Due to the rapid evolution
  
  • Bacteria may be resistant to one or several types of anti-microbial agents

Question 8

Innate resistance- Exclusion

Answer 8

• Gram +Ve bacteria: single cell wall
• Gram -Ve bacteria: two cell walls + periplasm
• Gram +Ve bacteria: cell wall only excludes very large (>50 KD) molecules, so uptake of antibiotics is not impeded
• The outer membrane of Gram -Ve bacteria is a barrier to uptake of antibiotics

Answer 9

• B-lactams; Quinolones; Tetracyclines and macrolides diffuse across the outer membrane via porin channels
• Aminoglycosides promote their own passage through the outer membrane by binding to LPS

Question 10

Porins

Answer 10

• Porins are barrel-shaped transmembrane proteins that act as passive pores
• Loss through mutation can lead to resistance
  
  • Loss of the Pseudomonas aeruginosa oprD porin used by Imipenem an IV B-lactam anti-biotic increases MIC from 1-2 to 8-32 mg/L (17% rate of resistance reported)
  • No energy use

Question 11

Innate resistance- efflux

Answer 11

• Antibiotics cross the outer membrane via porin channels or by self-promoted uptake
• But are removed by energy-consuming efflux pumps before they can act on their targets

Question 12

Other mechanism

Answer 12

• Overproduce target
  
  • Target high enough that enough is unligated to function and for bacteria to survive
• Bypass inhibition
  
  • Changes to the substrate specificity of the enzyme to which an antibiotic does not bind- allows metabolism to continue

Question 13

Acquired resistance

Answer 13

• Bacterial population + antibiotic = mutation in bacteria = resistance
• Resistant bacteria fill ecological niche left by antibiotic-sensitive bacteria
• The resistant population then grow and thrive
• When you kill the susceptible bacteria and just leave resistant bacteria, the resistant strain can grow rapidly and fill the ecological niche

Question 14

Genetic mechanism of antibiotic resistance

Answer 14

• Transformation- direct uptake of DNA containing resistance genes, e.g. altered PBP’s in penicillin-resistant Strep.pneumonia
• Transposition- Movement of resistance genes from plasmid to genome e.g. VRE
• Mutation- base changes or deletions lead to altered gene expression e.g. DNA gyrase, DNA polymerase, chromosomal B-lactamase
• Conjugation- plasmids transferred from other organisms e.g. B-lactamase, AG adnCAT modifying enzymes, mecA (PBP2) in MRSA

Question 15

Mechanisms to resist antibiotic action

Answer 15

• Inactivation and modification
  
  • Destroy antibiotic through chemical modification (changes the antibiotic structure so it does bind) (horizontal)
• Changes in the specific target protein
  
  • Reduce antibiotic binding to the target (Changes target structure so antibiotic does not bind) (Vertical)
• Microbes use both mechanisms to resist antibiotic action

Question 16

Inactivation and modification enzyme

Answer 16

• B-lactamases: over 300 different enzymes identified to date, including penicillinases, cephalosporinases, carbapenemases
  
  • Clavulanic acid, sulbactam and tazobactam developed as inhibitors
  • Extended-spectrum enzymes now common, inhibitor-resistance also described but rare
• Aminoglycoside-modifying enzymes
  
  • Acetylation, phosphorylation, adenylation reaction
  • Amikacin developed to block one common enzyme
• Chloramphenicol acetyltransferase
  
  • Common in salmonella where chloramphenicol used OTC

Question 17

Changes in target enzymes or substrates

Answer 17

• Penicillin-binding proteins: MecA in MRSA
• DNA gyrase (gyrA, gyrB): quinolones
• Dihydrofolate reductase: trimethoprim
• 23 SrRNA methylation: erythromycin
• RNA polymerase: Rifampicin
• D-ala-D-ala to D-ala-D-lac: VRE
• Peptidoglycan thickness and reduced glutamate amidation: VISA, GISA

Question 18

Approaches to counter antibiotic resistance

Answer 18

• Vaccine
• Bacteriophages
• New antibiotics

Question 19

Vaccine

Answer 19

• T cell or Ab-mediated
• Attenuated whole organism
• HHeat-inactivated whole organism
• Chemically inactived whole organism
• Sub-unit or toxoid
• carbohydrate epitope
• • lots of experiemental vaccine(peptide)
• Often need adjuvants (alum, SMA, saponins)
• Many ways of delivering vaccines (raw DNA, viral vectors, liposomes)
• No issue with resistance. Once a lifetime to once a year dosing-

Question 20

Bacteriophages

Answer 20

• Viruses that infect and destroy bacteria

1. Binding
2. Injection of nucleic acid
3. Re-programming
4. Production of phage particles
5. Assembly
6. Lysin, lysis and escape

Question 21

Bacteriocins and Antimicrobial peptides (or host defence peptides)

Answer 21

• Bacteriocins- proteins or peptide antibiotics secreted by bacteria to disadvantage other bacteria
• Antimicrobial peptides
• Host defence peptides- part of innate immunity (immune system peptides)

Question 22

Antibiotics to counter antibiotic resistance

Answer 22

1. Use existing antibiotics more carefully
   
   • Antibiotic policies, biocides, hand-washing
2. Develop existing agents
3. Target resistance mechanism
   
   • B-lactamase inhibitors (clavulanic acid, sulbactam, tazobactam) or efflux pump blockers (glycylcyclines, tigecycline)
4. Develop agents against new targets
   
   • Topoisomerase IV (alternate DNA gyrase), DNA polymerase, tRNA synthetases, peptide deformylase, membrane-active peptides

• Bacterial genomics can suggest lots of targets

Question 23

Antibiotic targets

Answer 23

• DNA-directed RNA polymerase= Rifampicin
• DNA gyrase= Quinolones
• Folic acid metabolism= Sulphonamides, Trimethoprim
• Protein synthesis
  
  • 50S = Macrolide; Chloramphenicol; Clindamycin
  • 70S= Aminoglycosides; tetracycline
  • 50 + 30S complex= Oxazolidinones (linezolid)

Question 24

New agents with activity vs MRSA

Answer 24

• Linezolid = Oxazolidinones = MRSA and VRE
• Tigecycline = Efflux blocker
• Daptomycin = MRSA and VRE

Question 25

Other new drugs near to launch

Answer 25

• Arpida iclaprim (DHFR inhibitor)
• Pfizer Dalbacancin
• Astella telavancin
• Targanta oritavancin
• Forest and takeda ceftaroline
• Basilea and J&J ceftobiprole

Question 26

Fidaxomicin

Answer 26

• Fidaxomicin (Dificlir) is the first in a new class of macrocyclic antibiotics recently licensed by the European medicines agency (EMA) to treat Clostridium difficile infection (CDI)
• Trials suggest Fidaxomicin is not inferior to vancomycin for mild to severe CDI
• Fidaxomicin has not been compared to metronidazole in clinical trials
• Its side effect profile appears similar to that of oral vancomycin and it may have advantages in reducing the rate of recurrence

Question 27

New Antibiotic

Answer 27

• Active against the microbial target
• Kills bacteria or interferes with bacterial growth
• Does not kill ‘good’ bacteria
• Inactive against humans