Class 12: Antibiotic Resistance

1920s–1930s: Alexander Fleming observes a “dead zone” around a fungal contaminant (Penicillium) on a Petri dish ➜ birth of penicillin.
- Chain & Florey later isolate and mass-produce penicillin; all three share the Nobel Prize.
Antibiotics are 100 years old—an extremely recent innovation in medical history.

Explosive growth rate
- Optimal temperature in the body $\approx 37\, ^\circ\text{C}$ .
- Typical doubling time $\tau \approx 20\text{ min}$ .
- Population after time $t$ : $N = N_0 \times 2^{t/\tau}$ .
Toxin production
- Secreted toxins can overwhelm host tissues or immune responses.
Immune-system interference: High density + toxins = systemic damage.

Chromosome – essential genes.
Plasmid – extra-chromosomal DNA; often carries antibiotic-resistance genes.
Cell wall – peptidoglycan mesh providing rigidity.
Pilus, capsule – virulence/adhesion; not examinable in detail per instructor.

Classification based on uptake of a purple Gram stain.
Gram-positive
- Single phospholipid membrane.
- Thick peptidoglycan layer ➜ stain penetrates easily.
Gram-negative
- Two hydrophobic membranes (outer + inner) separated by a thin peptidoglycan layer.
- Outer membrane contains porins (protein channels) for nutrient uptake.
- Dual membranes create a formidable permeability barrier ➜ infections harder to treat.

Structural mimic of the peptide substrate ➜ binds transpeptidase active site.
Acts as an irreversible competitive inhibitor—forms covalent bond, permanently inactivating the enzyme.
Blocked peptidoglycan synthesis ⇒ bacterium cannot divide.

Cell-wall inhibitors: Penicillin, Vancomycin (block peptidoglycan assembly).
Sulfonamides ("sulfa drugs"): Inhibit folate biosynthesis pathway.
Protein-synthesis inhibitors (ribosome binders): Kanamycin, Chloramphenicol, Lincomycin.
- Lincomycin famously blocks chloroplast translation of D1 protein in photosynthesis labs.
DNA/RNA synthesis inhibitors: Ciprofloxacin (Cipro) targets DNA gyrase (topoisomerase acting on circular DNA).

No bacterial-type cell wall in humans.
Folate: Humans obtain it from diet; we lack the inhibited biosynthetic enzymes.
Ribosomes: Bacterial (70 S) ≠ Eukaryotic cytosolic (80 S) ➜ antibiotics discriminate.
DNA gyrase mainly acts on circular genomes—absent from human nuclei.
Mitochondrial exception
- Mitochondria retain prokaryotic ribosomes & DNA gyrase.
- Limited toxicity because:
  - Mitochondria divide slowly (≪ every 20 min).
  - Antibiotic courses are short (< 1 month); physicians rotate drugs if prolonged.

Penicillin introduced 1941; Staphylococcus aureus resistance detected within 1 year.
Ciprofloxacin (1987) saw resistance by ≈2007.
Pharma dilemma: $\approx\$200\,\text{M}$ & 10 years R&D ⇒ resistance often emerges within a decade.

Lawn of bacteria grown on agar; paper disks soaked with antibiotics placed on surface.
Clear “dead zones” = effective killing.
Small/no clearing ➜ high resistance (e.g., Disk B in lecture image).

Mutations
- Arise randomly during DNA replication.
- $\text{Most} (\approx 99.9\%)$ are deleterious or neutral.
- Extremely rare beneficial mutations (e.g., altered transpeptidase) become common due to:
- Short doubling time.
- Huge populations $10^8–10^{10}\;\text{cells mL}^{-1}$ .
Harvard MEGA-Plate Experiment
- Visualized stepwise evolution to withstand $10^3$ -fold antibiotic concentration in ~11 days.

Donor cell extends sex pilus / conjugation tube.
Plasmid (often harboring resistance genes) replicates & transfers to recipient.
Type of horizontal (lateral) gene transfer, accelerating spread of resistance.

Reduced uptake: Down-regulate or mutate porins ⇒ antibiotic cannot enter (intrinsic or mutated).
Target modification: Point mutation alters drug-binding site (e.g., altered transpeptidase).
Efflux pumps: Up-regulation of ABC transporters actively expels drug.
Bypass / Alternative pathway: Acquire or evolve novel enzyme performing same biochemical step via a different route.
- Example: New folate-synthesis enzyme insensitive to sulfonamides.

Discovered 2015 from environmental isolates.
Potent against multi-drug resistant Staphylococcus aureus.
Unique MoA
- Binds non-protein targets lipid II & lipid III (glycolipid precursors).
- Simultaneously blocks two independent pathways:
1. Peptidoglycan synthesis.
2. Wall teichoic-acid synthesis.
Advantages
- Targets are not proteins ⇒ mutational escape far less likely.
- Single molecule inhibits two pathways ⇒ dual hurdle for resistance.
Empirical data (first publication)
- Time-kill curves: Eliminates cultures within $\le 16\,\text{h}$ similar to current drugs.
- 25-day serial-passage experiment: No increase in minimum inhibitory concentration (MIC); traditional drugs showed stepwise MIC elevation.

Evolutionary principles (random mutation + selection) are directly observable in microbial timescales.
Overuse/misuse of antibiotics in medicine & agriculture accelerates selection for resistance.
Economic challenge: high R&D cost vs. fast obsolescence disincentivizes pharmaceutical investment.
Ethical imperative: stewardship—prescribing appropriate doses/durations, surveillance, and development of novel drugs.

Doubling time $\tau = 20\,\text{min}$ .
Population scale: $10^8–10^{10}\,\text{cells mL}^{-1}$ .
MEGA-plate antibiotic zones: $1×,\;10×,\;100×,\;1000×$ concentration gradient.
Mutation impact frequency: beneficial $\sim 10^{-4}\text{–}10^{-5}$ relative to harmful/neutral.
Pharma development: $\$2\times10^8$ cost, > 10 years.

Be able to match EACH antibiotic class with its molecular target.
Recognize why selective toxicity stems from structural/biochemical differences.
Distinguish intrinsic vs acquired resistance mechanisms.
Practice interpreting disk-diffusion plates & MIC graphs.
Be prepared for conceptual questions on evolution (mutation + selection) in microbial populations.