Antimicrobial Chemotherapy – Key Vocabulary

Introduction to Antimicrobial Chemotherapy

• Definition & Aim

  • Use of chemicals to treat infectious disease.

  • Core principle: selective (differential) toxicity – agents must be more toxic to microbes than to the human host.

  • Achieved by targeting structures/processes absent or very different in eukaryotic cells (e.g. peptidoglycan, bacterial ribosome sub-sites, DNA gyrase).

• Types of Antimicrobial Chemicals

  • Disinfectants: high toxicity/corrosive; used on inanimate surfaces (e.g. hypochlorite bleach).

  • Antiseptics: significant systemic toxicity; safe for topical use (e.g. benzalkonium chloride).

  • Antibiotics / Antimicrobial drugs: safe for systemic administration (e.g. penicillin). Same molecule can fall into different categories at different concentrations.

• Bactericidal vs Bacteriostatic

  • Bactericidal: kill bacteria outright (e.g. \beta–lactams); preferred, essential for endocarditis/meningitis where host defence is weak.

  • Bacteriostatic: inhibit growth; immune system must clear infection (e.g. tetracyclines, macrolides).

• Spectrum of Activity

  • Narrow spectrum: limited taxa.

  • Broad spectrum: wide range (useful empirically, but disrupts normal flora).

• The Arms Race (Historical Perspective)

  • Term “Modern Chemotherapy” coined by Paul Ehrlich (1908).

  • Continuous cycle: discovery/deployment of new drugs ⇆ evolution/acquisition of resistance genes.

• Sources of Drugs

  • Synthetic: lab-made small molecules (e.g. sulphonamides).

  • Natural (Antibiotics): microbial secondary metabolites (e.g. penicillin from Penicillium).

  • Semi-synthetic: chemically modified natural scaffolds (many \beta–lactams, tetracyclines).

• Waksman’s 1941 Definition

  • “Chemical substances produced by microorganisms that, in dilute solution, selectively inhibit or destroy other microorganisms.”

  • Dilute-solution clause highlights high potency & selective toxicity.

Molecular Targets & Mechanisms of Action

• Major Target Classes

  • Cell-wall biosynthesis

  • Cytoplasmic membrane function

  • Nucleic-acid synthesis (DNA replication / transcription)

  • Protein synthesis (ribosome)

  • Key metabolic pathways (e.g. folate synthesis)

Cell-Wall Biosynthesis Inhibitors

• Peptidoglycan (PG) essentials

  • Unique to bacteria → excellent selective target.

  • Consists of \text{NAG–NAM} glycan chains cross-linked via peptide side-chains.

  • Two critical enzyme classes:

  • Transglycosylases – build glycan backbone.

  • Transpeptidases (Penicillin-Binding Proteins, PBPs) – cross-link peptides.

1. \beta–Lactam Antibiotics

• Core \beta-lactam ring conserved; side chains tune spectrum, stability, resistance profile.

• Classes & Notable Examples

  • Penicillins: benzylpenicillin (G), amoxicillin, flucloxacillin (anti-staph).

  • Cephalosporins (1st→5th gen): cephalexin → ceftaroline (MRSA-active).

  • Carbapenems: meropenem, ertapenem (very broad, last-resort).

  • Monobactams: aztreonam (Gram-negative aerobes).

• Mechanism

  • Structural mimic of terminal D\text{-Ala–D-Ala} dipeptide in PG precursors.

  • Act as suicide substrates for PBPs → covalent acyl-enzyme complex → irreversible inhibition → failure to cross-link → cell lysis.

  • Bactericidal; different \beta-lactams have varying PBP specificities.

2. Glycopeptides – Vancomycin

• Large molecule (C${66}$ H${74}$ Cl N$9$ O${24}$) from Actinomycetes.

• Binds directly to D\text{-Ala–D-Ala} termini of lipid II & uncross-linked PG → blocks both transglycosylation & transpeptidation.

• Active only on Gram-positives (too big to cross Gram-negative outer membrane).

3. Membrane Disruptors – Polymyxins (e.g. Colistin)

• Cyclic cationic peptides; act like detergents.

• Target anionic LPS in Gram-negative outer membrane; displace Mg^{2+}/Ca^{2+} → permeability, lysis.

• Limited by nephro- & neuro-toxicity; topical or IV for MDR infections; not absorbed orally.

Nucleic-Acid Synthesis Inhibitors

1. Transcription – Rifampicin

• Binds \beta-subunit of bacterial RNA polymerase (RNAP) → blocks promoter clearance/elongation.

• Essential drug (in combination) against Mycobacterium tuberculosis.

2. DNA Replication – Quinolones/Fluoroquinolones

• Bicyclic 4-quinolone core (e.g. nalidixic acid, ciprofloxacin).

• Inhibit DNA gyrase & Topo IV → prevent relaxation of positive supercoils ahead of replication fork → bactericidal.

• DNA Topology Refresher

  • Negative supercoiling (under-wound) facilitates strand separation.

  • DNA gyrase introduces negative coils; inhibition stalls replication.

Protein Synthesis Inhibitors (Ribosome)

• Bacterial ribosome 70S = 30S + 50S; distinct from human 80S → high selectivity.

Metabolic Inhibitors – Sulphonamides & Trimethoprim (Homework I)

• Target folate synthesis (absent in humans who obtain folate via diet).

• Sulphonamides inhibit dihydropteroate synthase; trimethoprim inhibits dihydrofolate reductase ➔ sequential blockade → synergistic (bactericidal combination).

Antibiotic / Antimicrobial Resistance (AMR)

• Definition: heritable reduction in susceptibility encoded by resistance genes (plasmid or chromosome).

• Historical Warnings & Timeline

  • Fleming (1945): foresaw resistance.

  • Penicillin deployed 1943 ➔ resistance in 1946.

  • Similar rapid emergence for almost every new class (see list: sulphonamides – 1940s, methicillin – 1961, etc.).

• Mechanistic Categories

  1. Exclusion

  • ↓ Uptake (porin loss in Gram-negatives).

  • ↑ Efflux pumps.

  1. Drug Inactivation

  • Enzymatic hydrolysis (e.g. \beta-lactamases).

  • Chemical modification (acetylation, phosphorylation).

  1. Target Modification

  • Point mutations (e.g. gyrA in quinolone resistance).

  • Replacement enzymes (e.g. mecA PBP2a in MRSA).

• \beta-Lactamases

  • Evolved from ancestral PBPs (structural similarity).

  • TEM-1 hydrolyses penicillins; ESBLs & carbapenemases (e.g. NDM-1) broaden spectrum.

• Evolutionary Drivers

  • Natural selection under antibiotic pressure.

  • Horizontal Gene Transfer: transformation, transduction, conjugation (plasmids/transposons).

  • Some resistance determinants are ancient (e.g. van genes in 30 000-year permafrost) – antibiotics are natural ecological weapons.

• Multidrug Resistance (MDR)

  • Co-localised gene cassettes/plasmids (e.g. 180-kb NDM-1 plasmid) ➔ strains untreatable except with toxic agents like colistin.

• Global Threat

  • No new antibiotic classes since 1987; O’Neil Review projects \approx 10^7 AMR-associated deaths annually by 2050.

• Mitigations

  • Discovery of new classes/alternatives (phage therapy).

  • Antibiotic stewardship (prudent use, diagnostics, surveillance).

Antimicrobial Susceptibility Testing (AST)

• Role in Stewardship: guides effective therapy, reduces empirical over-use.

• Requires rapid, accurate ID + susceptibility profile.

Qualitative Method – Kirby–Bauer Disc Diffusion

• Paper discs with fixed antibiotic concentration on agar lawn.

• Diffusion creates decreasing radial gradient; measure zone of inhibition.

• Standardised variables (BSAC/EUCAST):

  • Inoculum density (0.5 McFarland).

  • Medium (Mueller–Hinton/Iso-Sensitest; PABA-free so sulphonamides remain active).

  • Agar depth (affects diffusion rate).

  • Disc potency, incubation time.

• Direct testing possible on urine/blood in emergencies.

• Interpretation via zone-diameter breakpoints (susceptible/intermediate/resistant).

Quantitative Concept – Minimum Inhibitory Concentration (MIC)

• Definition: lowest [drug] preventing visible growth.

• Organism-drug specific; expressed in \text{mg L}^{-1}.

• MIC \le CLSI/EUCAST breakpoint ⇒ “susceptible”.

Determination Methods

1. E-test (Gradient strip) – logarithmic antibiotic gradient; MIC read where ellipse intersects strip.

2. Broth Dilution (Gold Standard) – doubling series in microtitre; turbidity readout.

3. Automated Systems – VITEK, Phoenix: monitor growth curves, extrapolate MIC; integrated ID.

• Minimum Bactericidal Concentration (MBC)

  • Lowest [drug] killing \ge 99.9\% of initial inoculum.

  • Determined by sub-culturing MIC wells onto drug-free agar.

  • Bactericidal agents: MBC ≈ MIC.

  • Bacteriostatic: MBC much higher (≥ 4× MIC).

  • Crucial for treating CNS infections or immunocompromised patients where host clearance is poor.

Learning Outcomes Recap

• Identify key molecular targets: cell wall ( \beta-lactams, vancomycin ), DNA synthesis (quinolones), protein synthesis (aminoglycosides, tetracyclines, macrolides ).

• Explain main AMR mechanisms, with \beta-lactamases as exemplar; appreciate evolutionary & ecological origins.

• Perform & interpret AST: disc diffusion, MIC, MBC; understand clinical breakpoints & stewardship impact.

Worked Example – MIC/MBC (from Slides 70-71)

• Broth dilution results: growth in tubes \le 8 mg L$^{-1}$; clear at \ge 16 mg L$^{-1}$.

  • \text{MIC} = 16\,\text{mg L}^{-1}.

• Sub-culture shows no colonies (bactericidal) at 64 mg L$^{-1}$, growth at 32 mg L$^{-1}$.

  • \text{MBC} = 64\,\text{mg L}^{-1}.

• Since \text{MBC}/\text{MIC} = 4, borderline static ↔ cidal; generally interpreted as bactericidal if \le 4.

Connections & Real-World Relevance

• Concepts connect back to earlier microbiology (cell envelope, genetics) & forward to clinical practice (choice of empiric therapy, infection control).

• Ethical/policy dimension: balancing life-saving drug use vs resistance emergence; global stewardship initiatives; O’Neil economic modelling.

• Practical implications: lab selection of media, need for rapid diagnostics, toxicity management when colistin is last option.