9 - Mode of Action

• Antimicrobial resistance is more prevalent in Africa, the middle east and South America
• Biofilm - an assembly of microbial cells associated with a surface and enclosed in an extracellular matrix made principally of polysaccharides (extracellular polymeric substance (EPS) matrix).
• How a Biofilm forms:

  Motile cells stick to a surface, they differentiate into matrix producers then grow and aggregate before reaching maturation, forming spores and dispersing.
• Habitat of Biofilms:

  localised gradients, sorption for resource capture, enzyme retention for an external digestion system, cooperation, competition and tolerance and resistance.
• Biofilm resistance:

  Slow diffusion leads to tolerance

  Sublethal concentrations of antimicrobials in the biofilm leads to selection of antimicrobial resistance (AMR)

  These resistant genes are then transferred
• Antibiotic

  Any natural, semisynthetic, synthetic product that has the ability to kill or inhibit the growth of bacteria.
• Antimicrobial

  Any product that has the ability to kill or inhibit the growth of microorganism including: bacteria, viruses, fungi
• Antibiotic resistance mechanisms

  Gram negative - intrinsically resistant due to a double membrane structure making the cellular envelope relatively impermeable

  Gram positive - acquired resistance due to alterations to envelope structure such as porin loss
• Non-specific antibiotic resistance mechanisms:

  under expression of porins and accumulation of efflux pumps
• Specific antibiotic resistance:

  Inactivation of antibiotic via an antibiotic modifying enzyme

  Target site modification

  Target protection via a target protection protein

  Target bypass - new proteins with same metabolic capacity as antibiotic target protein
• Antibiotics that target cell wall synthesis:

  • penicillins
  • cephalosporins
  • carbapenems
  • monobactams
  • glycopeptides
  • vancomycin
• Antibiotics that inhibit nucleic acid synthesis

  • quinolones
• Antibiotics that inhibit protein synthesis:

  30s subunit (first 2) 50s subunit (last 2)

  • aminoglycosides
  • tetracyclines
  • macrolides
  • chloramphenicol
• Rifamycins inhibit RNA synthesis
• polymixins disrupt the cell membrane
• sulfonamides inhibit folate synthesis
• The common group of antibiotics that inhibit cell wall synthesis

  
• Most β-lactam antibiotics target penicillin-binding proteins
• Penicillin-binding proteins (PBPs) are DD-transpeptidases that make cross-links (peptide bonds) between D-amino acid residues in sugar-linked pentapeptides in the bacterial cell walls.

  PBPs are essential for bacterial cell wall synthesis.
• The reactive β-lactam ring binds the active site of the transpeptidase, permanently inactivating the PBP:  cell wall cross-linking ceases
• Resistance mechanisms to B-lactam antibiotics

  ① mutation of PBP, lowering the affinity for penicillins, etc

  ② down-regulation of porins in Gram-negative bacteria;

  ③ acquisition of B-lactamase- ESBL (extended spectrum β-lactamase);

  ④ up-regulation of efflux pumps
• The common group of antibiotics that inhibit nucleic acid synthesis

  
• A quinolone antibiotic is a member of a large group of broad-spectrum bacteriocidals that share a bicyclic core structure related to the compound 4-quinolone.
• Quinolone antibiotics interfere with nucleic acid synthesis by binding to topoisomerases required for unwinding of DNA.
• Gyrase and topoisomerase IV are heterotetrametric enzymes that consist of two A subunits and two B subunits:

  B subunit carries out the nicking of DNA,

  A subunit introduces negative supercoils,

  and then reseals and ligates the strands.
• Quinolones act by converting their targets, type II topoisomerases (gyrase and topoisomerase IV), into toxic enzymes that fragment the bacterial chromosome.

  These double stranded cuts are hard to repair.

  Ultimately they cause bacterial cell death.
• Quinolones bind gyrA. Gyr B can still make the ds cut but the loop can no longer be passed through the cut so the DNA is cut but cannot be ligated by topo II.
• Resistance mechanisms to quinolones:

  ① Mutations in gyrase and topo IV weaken quinolone-enzyme interactions

  ② Plasmid encoded Qnr proteins decrease topoisomerase-DNA binding

  ③ A plasmid encoded enzyme acetylates ciprofloxacin, decreasing its effectiveness.

  ④ Plasmid encoded efflux pumps decrease the concentration of quinolones in the cell.
• Antibiotic protein synthesis inhibitors 50S:

  • Macrolides effect translocation e.g. erythromycin
  • Chloramphenicol binds to the 50S ribosomal subunit and inhibits the formation of peptide bonds
• Antibiotic protein synthesis inhibitors 30S:

  • Tetracycline binds to the 30S subunit and interferes with the binding of tRNA to the ribosomal complex
  • Aminoglycosides bind to the 30S subunit and cause codon misreading:  some interfere with formation of the initiation complex. e.g. streptomycin, kanamycin, gentamicin
• Aminoglycoside shared structures

  An aminoglycoside antibiotic contains amino-sugar structures

  
• Aminoglycosides display bactericidal activity against Gram-negative aerobes but generally not against anaerobic Gram-negative bacteria.

  Streptomycin is an example of an aminoglycoside.
• 1. Aminoglycosides inhibit protein synthesis by high affinity binding to the A-site on the 16S ribosomal RNA of the 30S ribosome.
  2. Codon misreading.  This leads to misincorporation of amino acids – error prone protein synthesis
  3. This can cause some of the protein products to MISFOLD
  4. If misfolded membrane proteins are incorporated into the cell envelope, the membranes become leaky, leading to increased drug uptake. This leads to rapid uptake of more aminoglycoside molecules into the cytoplasm and increased inhibition of protein synthesis and mistranslation
• Aminoglycosides have synergism with antibiotics such as β-lactam

  Allows greater penetration of Aminoglycosides at low dosages

  All aminoglycoside antibiotics are rapidly bactericidal to Gram-negative bacteria in aerobic conditions.
• Resistance to Aminoglycosides

  • Aminoglycosides can be modified by acetyltransferases
  • Methylation of the 16S rRNA by ribosomal methyltransferase prevent Aminoglycosides from binding to this target