L04 I&I Intro to Antibiotics

Overview

This lecture introduces the major classes of antibiotics, organised by the bacterial process each targets: cell wall synthesis, the cell membrane, folate synthesis, nucleic acid synthesis, and protein synthesis. For each class it covers the drugs involved, spectrum (Gram-positive vs Gram-negative), and the specific molecular mechanism of action, using a recurring “roadmap” diagram of a bacterial cell to map drug class to target site. It closes with clinical spectrum charts and a historical view of how quickly resistance has followed the deployment of each antibiotic class.

Why antibiotics matter

• Comparison of Staphylococcus aureus bacteraemia outcomes: 1941 mortality rate 82% versus 2009 mortality rate 21%, illustrating the impact of antibiotics on a previously often-fatal infection.
• In the 1941 cohort (122 cases), the number of deaths closely tracked the total number of cases across nearly all age bands, i.e. survival was uncommon. In the 2009 cohort, survivors substantially outnumbered deaths in every age band, with case numbers highest in the 60-89 age range.

What antibiotics are

• Antibiotics inhibit bacterial growth.
• They work by interfering with specific bacterial enzymes.
• Differential toxicity for bacterial over human (eukaryotic) cells depends on one of two conditions:
  • the drug’s target is not present in eukaryotic cells, or
  • the target is sufficiently different in eukaryotic cells.

Bacterial cell wall structure

• Gram-positive cell wall: a thick, multilayer peptidoglycan mesh anchored by lipoteichoic acid (LTA), sitting directly on the cytoplasmic membrane.
• Gram-negative cell wall: a thin peptidoglycan layer confined to the periplasmic space between two membranes; the outer membrane is studded with lipopolysaccharide (LPS), protein and porins, and a lipoprotein tethers the peptidoglycan to the inner membrane.
• This structural difference underlies why some antibiotics (e.g. glycopeptides, some polymyxins/daptomycin) are restricted to Gram-positive or Gram-negative organisms only.

Cell wall synthesis inhibitors

Beta-lactams

• Classes: penicillins, cephalosporins, carbapenems, monobactams. All share a four-membered beta-lactam ring, fused to different core ring structures (or, for monobactams, present alone).
• Multiple drugs exist within each class due to modification of R side chains, which affects spectrum, resistance to beta-lactamases, bioavailability and pharmacokinetics.
• Normal peptidoglycan cross-linking: a penicillin-binding protein (transpeptidase) engages the pentaglycine chain of one peptidoglycan strand’s peptide side chain (built on a NAM/NAG backbone with alanine, D-glutamate and L-lysine residues) and links it to the peptide side chain of an adjacent strand, releasing a terminal alanine residue in the process, producing cross-linked peptidoglycan.
• Mechanism of beta-lactams: they bind irreversibly to the penicillin-binding protein, forming a covalent bond with a serine residue in its active site. This occupies the enzyme so it cannot engage its normal substrate, inhibiting cross-linking of peptidoglycan peptide side chains, the last step in peptidoglycan synthesis.
• Allergy: most commonly reported against penicillins, but over-reported, 95% of patients with a history of penicillin allergy are found not to be truly allergic on testing. The relevant allergen may be the shared beta-lactam ring or the drug-specific R side chain. Cross-reactivity of penicillin allergy is approximately 2.5% with cephalosporins and under 1% with carbapenems and monobactams.

Glycopeptides

• Drugs: vancomycin, teicoplanin.
• Spectrum: Gram-positive only, as they are unable to penetrate the Gram-negative outer membrane.
• Vancomycin was isolated in 1953 at Eli Lilly from a soil sample collected in the interior jungles of Borneo, produced by Amycolatopsis orientalis.
• Mechanism: vancomycin binds directly to the terminal D-alanine-D-alanine portion of the peptidoglycan pentapeptide side chain (muramylpentapeptide) before the cross-linking enzyme can act. The cell-wall cross-linking enzyme is then sterically blocked from engaging the peptide chain, so the strands remain unlinked. This inhibits cross-linking by steric blockade of the substrate, in contrast to beta-lactams, which inhibit by binding the enzyme itself.

Cell membrane-acting antibiotics

Polymyxins

• Drugs: colistin, polymyxin B. Cyclic cationic lipopeptides.
• Spectrum: Gram-negative only (some species are intrinsically resistant).
• Mechanism: electrostatic interaction with LPS in the outer membrane. Sequence: colistin binds and disrupts the outer LPS leaflet, displacing Ca2+ and Mg2+; it then crosses into the periplasm and disrupts the inner phospholipid membrane, causing cell lysis.

Daptomycin

• Cyclic lipopeptide.
• Spectrum: Gram-positive only, dependent on the phospholipid content of the cell membrane; binds to phosphatidylglycerol in the cell membrane.
• Mechanism: binds calcium at the outer leaflet of the phospholipid bilayer, inducing membrane curvature, then oligomerises to form an ion channel spanning the bilayer, permeabilising the membrane.

Folate synthesis inhibitors

• Target the bacterial pathway that produces tetrahydrofolic acid (FH4), an essential co-factor for nucleic acid synthesis.
• Pathway: pteridine + p-aminobenzoic acid to dihydropteroic acid, plus n-glutamic acid to dihydrofolic acid (FH2), then via dihydrofolate reductase to tetrahydrofolic acid (FH4).
• Sulphonamides: structurally similar to p-aminobenzoic acid; competitively inhibit the first step of the pathway.
• Trimethoprim: structurally similar to dihydrofolic acid; competitively inhibits dihydrofolate reductase (the FH2 to FH4 step).
• Sulphonamides and trimethoprim therefore block two sequential steps of the same folate synthesis pathway.

Nucleic acid synthesis inhibitors

Fluoroquinolones

• Inhibit type II topoisomerases (DNA gyrase and topoisomerase IV), which inhibits bacterial DNA replication.
• Type II topoisomerases normally introduce negative supercoils: they unwind over-twisted (positively supercoiled) DNA into a relaxed state, converting it toward the negatively supercoiled state required to package DNA into the bacterial cell, cycling between positive supercoils (generated ahead of the replication fork), relaxed DNA, and negative supercoils.
• Example drug: ciprofloxacin.
• Resistance to quinolones can develop by chromosomal mutation during therapy.

Rifampicin

• Inhibits RNA synthesis; its target is RNA polymerase.
• Resistance arises from a single point mutation and can develop by chromosomal mutation during therapy.

Metronidazole

• Makes breaks in DNA.
• A prodrug requiring activation, achieved by reduction via ferredoxin or flavodoxin.
• Ferredoxin and flavodoxin are the electron acceptors used by anaerobes and microaerophiles (pyruvate to acetyl-CoA via pyruvate:ferredoxin oxidoreductase, using ferredoxin and thiamine pyrophosphate), whereas aerobes and mitochondria instead use NAD (pyruvate to acetyl-CoA via pyruvate dehydrogenase).
• Because activation depends on ferredoxin/flavodoxin, metronidazole is selectively activated in anaerobic and microaerophilic organisms, not in aerobes or host mitochondria.

Protein synthesis inhibitors

• Bind to 16S or 23S rRNA within the ribosome’s functional sites; selectivity for bacteria arises from differences between prokaryotic and eukaryotic ribosomes.
• 16S rRNA, in the 30S subunit: targeted by aminoglycosides (e.g. gentamicin, streptomycin) and tetracyclines. Structural mapping shows 30S-subunit-binding drugs cluster near the mRNA decoding site (also including spectinomycin, kasugamycin, pactamycin, edeine).
• 23S rRNA, in the 50S subunit: targeted by macrolides (e.g. erythromycin), lincosamides (clindamycin), oxazolidinones (linezolid), and also chloramphenicol and streptogramins. Structural mapping shows 50S-subunit-binding drugs cluster near the peptidyl transferase centre and peptide exit tunnel (also including blasticidin S, sparsomycin, puromycin).

Clinical spectrum of activity

• A detailed understanding of antimicrobial spectrum is required for clinical practice (flagged as not required for ELM exams). Spectrum is described against Gram-positive cocci (MRSA, MSSA, streptococci), Gram-negative bacilli (E. coli, Klebsiella/Proteus, Pseudomonas, ESCAPPM), and anaerobes. MSSA = methicillin-susceptible S. aureus.
• Beta-lactam spectrum:
  • Penicillin and amoxicillin cover Gram-positive cocci only.
  • Aztreonam (a monobactam) covers Gram-negative bacilli only.
  • Meropenem/imipenem cover almost all groups, including anaerobes; ertapenem is similarly broad but does not cover Pseudomonas.
  • Cephalosporin generations: 1st generation (e.g. cefazolin), good Gram-positive plus some Gram-negative activity; 2nd generation (e.g. cefuroxime), better Gram-negative activity; 3rd generation (e.g. ceftriaxone, cefotaxime, ceftazidime), improved stability against Gram-negative beta-lactamases, with or without Pseudomonas cover; 4th generation (e.g. cefepime), covers AmpC-producing organisms and Pseudomonas.
  • Enterococcus spp. and Listeria spp. are intrinsically resistant to cephalosporins.
• Non-beta-lactam spectrum: vancomycin and daptomycin, Gram-positive only; colistin, Gram-negative only with variable coverage (e.g. against ESCAPPM); tetracycline, mainly Gram-positive with some variable Gram-negative/anaerobe coverage; clindamycin, Gram-positive plus anaerobes; erythromycin and linezolid, Gram-positive; aminoglycosides, broad Gram-negative; trimethoprim and co-trimoxazole, Gram-positive plus Gram-negative (co-trimoxazole coverage partly variable); ciprofloxacin and moxifloxacin, broad Gram-positive through Gram-negative, with moxifloxacin also covering anaerobes; rifampicin, Gram-positive only with a limited role (not used as monotherapy); metronidazole, anaerobes only.

Antibiotic resistance timeline

• Across antibiotic classes deployed clinically between 1930 and 2005 (sulfonamides, penicillin, streptomycin, chloramphenicol, tetracycline, erythromycin, vancomycin, methicillin, ampicillin, cephalosporins, linezolid, daptomycin), resistance was, for most drugs, first observed only a few years after clinical deployment.
• This illustrates a historical pattern: resistance tends to emerge soon after each new antibiotic class enters clinical use.

Self-test

1. What three features define how antibiotics act on bacteria?
2. What two conditions give an antibiotic differential toxicity for bacterial over human cells?
3. Describe the key structural differences between the Gram-positive and Gram-negative bacterial cell wall.
4. What did the comparison of 1941 and 2009 S. aureus bacteraemia data show?
5. Describe the steps by which a penicillin-binding protein normally cross-links peptidoglycan.
6. Describe the mechanism by which beta-lactams inhibit peptidoglycan cross-linking.
7. Distinguish the mechanism of vancomycin from that of beta-lactams in inhibiting peptidoglycan cross-linking, and explain why vancomycin is Gram-positive only.
8. What proportion of patients with a reported penicillin allergy are found not to be truly allergic on testing, and what are the approximate cross-reactivity rates with cephalosporins versus carbapenems/monobactams?
9. Describe the steps by which colistin kills Gram-negative bacteria.
10. Describe the mechanism of daptomycin, including its calcium dependence.
11. List the two bacterial folate-synthesis steps blocked by sulphonamides and trimethoprim respectively, and state what each drug structurally resembles.
12. Describe the mechanism of action of fluoroquinolones, including the normal function of type II topoisomerases.
13. Distinguish the mechanism of rifampicin from that of fluoroquinolones.
14. Explain why metronidazole is selectively active against anaerobes and microaerophiles but not against aerobes or host mitochondria.
15. Distinguish the ribosomal targets and drug classes of 30S-subunit versus 50S-subunit protein synthesis inhibitors.
16. A patient with a presumed Gram-positive infection due to Enterococcus is prescribed cefazolin. Explain why this will not be effective.
17. Distinguish the spectrum coverage of meropenem from that of aztreonam.
18. What historical pattern is shown by the antibiotic deployment versus resistance-observed timeline (1930-2005)?
19. Integrative: a drug is found to bind irreversibly and covalently to a serine residue of an enzyme that normally cross-links peptide side chains of peptidoglycan. Identify the drug class, its target, and the resulting effect on the bacterial cell wall.

Answers

Reveal answers

1. Antibiotics inhibit bacterial growth, interfere with specific bacterial enzymes, and act with differential toxicity for bacterial over human cells.
2. Either the drug’s target is not present in eukaryotic cells, or the target is sufficiently different in eukaryotic cells.
3. Gram-positive: a single thick, multilayer peptidoglycan mesh anchored by lipoteichoic acid, sitting on the cytoplasmic membrane. Gram-negative: a thin peptidoglycan layer confined to the periplasmic space between an outer membrane (bearing LPS, protein, porins) and an inner membrane, with a lipoprotein tethering the peptidoglycan to the inner membrane.
4. Mortality from S. aureus bacteraemia fell from 82% in 1941 to 21% in 2009; in 1941 deaths closely tracked total cases across age bands, while in 2009 survivors substantially outnumbered deaths in every age band.
5. The enzyme engages the pentaglycine chain of one peptidoglycan strand’s peptide side chain and links it to the peptide side chain of an adjacent strand, releasing a terminal alanine residue, producing cross-linked peptidoglycan.
6. Beta-lactams bind irreversibly to the penicillin-binding protein, forming a covalent bond with a serine residue in its active site, occupying the enzyme so it cannot cross-link peptidoglycan peptide side chains.
7. Vancomycin binds directly to the terminal D-alanine-D-alanine of the peptide side chain, sterically blocking the cross-linking enzyme from engaging its substrate (blocking the substrate, not the enzyme, unlike beta-lactams). It is Gram-positive only because it cannot penetrate the Gram-negative outer membrane to reach the peptidoglycan.
8. 95% of patients with a reported penicillin allergy are found not to be truly allergic on testing. Cross-reactivity is approximately 2.5% with cephalosporins and under 1% with carbapenems and monobactams.
9. Colistin binds electrostatically to LPS and disrupts the outer LPS leaflet (displacing Ca2+/Mg2+), crosses into the periplasm, then disrupts the inner phospholipid membrane, causing cell lysis.
10. Daptomycin binds calcium at the outer leaflet of the phospholipid bilayer, inducing membrane curvature, then oligomerises to form an ion channel spanning the bilayer, permeabilising the membrane.
11. Sulphonamides block the first step (formation of dihydropteroic acid from pteridine and p-aminobenzoic acid) and structurally resemble p-aminobenzoic acid. Trimethoprim blocks the dihydrofolate reductase step (FH2 to FH4) and structurally resembles dihydrofolic acid.
12. Fluoroquinolones inhibit type II topoisomerases (DNA gyrase, topoisomerase IV), which normally unwind over-twisted DNA into a relaxed state and convert it to the negatively supercoiled state needed to package DNA into the cell; blocking this inhibits bacterial DNA replication.
13. Rifampicin inhibits RNA polymerase, blocking RNA synthesis, whereas fluoroquinolones inhibit type II topoisomerases, blocking DNA replication.
14. Metronidazole is a prodrug activated by reduction via ferredoxin or flavodoxin, the electron acceptors used by anaerobes and microaerophiles; aerobes and mitochondria use NAD instead, so the drug is not activated in these cells.
15. 30S subunit (16S rRNA): targeted by aminoglycosides and tetracyclines, binding near the mRNA decoding site. 50S subunit (23S rRNA): targeted by macrolides, lincosamides (clindamycin), oxazolidinones (linezolid), chloramphenicol and streptogramins, binding near the peptidyl transferase centre/peptide exit tunnel.
16. Enterococcus spp. are intrinsically resistant to cephalosporins, so cefazolin will not be effective regardless of the organism being Gram-positive.
17. Meropenem covers Gram-positive cocci, Gram-negative bacilli (including Pseudomonas) and anaerobes; aztreonam covers Gram-negative bacilli only.
18. For most drug classes, resistance was first observed only a few years after clinical deployment, showing that resistance tends to emerge soon after each new antibiotic class enters use.
19. This describes a beta-lactam antibiotic. Its target is the penicillin-binding protein (transpeptidase). The covalent binding occupies the enzyme’s active site, preventing cross-linking of peptidoglycan peptide side chains, the last step of peptidoglycan synthesis.