Antimicrobial Chemotherapy & Resistance – Core Vocabulary

Learning Outcomes

• Describe interplay between host immune defences, microorganisms, vaccination & immunotherapy.
• Perform lab isolation/diagnosis of pathogens; show problem-solving & data interpretation.
• Recognise commensal flora, microbial diversity in disease & select suitable diagnostics.
• Discuss principles of chemotherapy & resistance development at molecular, cellular & host levels.
• Explain pathogenic determinants shared across microbes and disease-specific pathogenesis.

Areas Covered in the Lecture

• Types of antimicrobial agents.
• Discovery & development of antibiotics.
• Key antibiotic properties & definitions.
• Bacterial cellular target sites.
• Antibiotic classes & their modes of action.
• Antimicrobial-resistance (AMR) mechanisms.

Antimicrobial Chemotherapy – Concepts

• Use of drugs to combat infectious agents: antibacterial, antifungal, antiparasitic, antiviral.
• Differential toxicity ⇒ drug harms pathogen more than host.

Antibiotics – Definition & Early History

• Antibiotic = naturally produced microbial substance that, in small amounts, inhibits/kills bacteria.
– Most current agents are natural scaffolds modified synthetically.
• Penicillin (Alexander Fleming, 1928) discovered serendipitously from Penicillium fungus.
– Limited funding until WWII (1942 mass-production in USA) → millions of lives saved.
• Pre-antibiotic chemotherapy relied on toxic metals (e.g. silver nitrate, arsenicals such as Salvarsan – \text{As=As} compound circa 1900).

Timeline of Key Discoveries

• 1900 Salvarsan (arsphenamine).
• 1940s Penicillins.
• 1940-60s "golden era" – streptomycin, tetracycline, chloramphenicol, etc.
• 1970-90s cephalosporins, carbapenems, quinolones, macrolides.
• 2015 Teixobactin (first‐in‐class; not yet marketed, expected ≤5 yrs).

Socio-Economic & Healthcare Impact

• Antibiotics underpinned modern surgery, transplantation, oncology, neonatal care, animal husbandry & global life-expectancy gains.

Natural Producers of Therapeutic Agents

• Gram-positive rods: Bacillus subtilis → bacitracin; B. polymyxa → polymyxin.
• Fungi: Penicillium notatum → penicillin; Cephalosporium spp. → cephalothin.
• Actinomycetes: Streptomyces venezuelae → chloramphenicol; S. griseus → streptomycin; S. nodosus → amphotericin B; Micromonospora purpurea → gentamicin.

What Makes an Ideal Antibiotic?

• Selective toxicity; cidal for pathogen; adequate tissue distribution; slow resistance development; oral & parenteral forms; minimal side-effects; cost-effective; compatible with other drugs.

Spectrum of Activity

• Narrow spectrum ⇒ active against limited taxa (e.g. penicillin G active mainly on Gram +; isoniazid specific to mycobacteria).
• Broad spectrum ⇒ active against wide diversity incl. both Gram + and – (e.g. tetracycline).

Key Quantitative Definitions

• Minimum Inhibitory Concentration (MIC): MIC = \min{C\,|\,\text{visible growth is inhibited}}.
• Minimum Bactericidal Concentration (MBC): MBC = \min{C\,|\, \ge99.9\%\,kill}.
• Bacteriostatic vs. bactericidal; time-dependent vs. concentration-dependent killing.
• Prophylaxis (pre-infection) vs. Treatment (established/suspected infection).

Bacterial Cell Structure & Its Therapeutic Relevance

• Cell wall (peptidoglycan of \text{NAG} & \text{NAM}) maintains shape, prevents osmotic lysis; absent in humans ⇒ prime target.
• Gram-positive ≈ thick PG layer + teichoic acids; Gram-negative ≈ outer membrane (LPS), thin PG, periplasm.
• Gram stain: crystal violet–iodine complex retained by Gram +, removed from Gram – (decolouriser then counter-stain safranin → pink).
• Plasma membrane, outer membrane (in –ve), ribosomes (70S), DNA gyrase, folate pathway, etc. are distinct from eukaryotic counterparts.

Antibacterial Target Sites & Mechanisms of Action

1. Inhibition of Cell-Wall Synthesis (bactericidal, time-dependent)

• β-Lactams: penicillins, cephalosporins, cephamycins, carbapenems.
– Bind Penicillin-Binding Proteins (PBPs) ⇒ block transpeptidation → weakened PG → lysis.
• Glycopeptides: vancomycin, teicoplanin (Gram + only).
– Bind D-Ala-D-Ala termini of nascent PG ⇒ prevent cross-linking.

2. Disruption of Cell Membrane Integrity

• Polypeptide antibiotics/antimicrobial peptides: polymyxin B, colistin; azole antifungals.
– Cationic molecules attracted to negatively charged LPS; insert into membrane → pores → leakage & death.

3. Inhibition of Nucleic-Acid Synthesis

• Quinolones/fluoroquinolones (ciprofloxacin, levofloxacin): inhibit DNA\ gyrase (Gram –) & topoisomerase\ IV (Gram +) ⇒ block DNA replication; concentration-dependent cidal.
• Rifamycins (rifampicin, rifabutin): bind bacterial RNA polymerase ⇒ halt transcription; cornerstone of anti-TB therapy.

4. Inhibition of Protein Synthesis (ribosomal blockers)

• Bind 30S or 50S subunits; usually broad spectrum.
– Macrolides (erythromycin), ketolides, lincosamides (clindamycin) – time-dependent static.
– Aminoglycosides (gentamicin) – irreversible 30S binding; concentration-dependent cidal.
– Tetracyclines – tRNA docking interference (30S).
– Chloramphenicol – peptidyl transferase inhibition (50S).
• Selective due to structural differences between pro- & eukaryotic ribosomes.

5. Antimetabolites / Folate-Pathway Inhibitors

• Trimethoprim + sulfamethoxazole (co-trimoxazole) block sequential steps in tetrahydrofolate synthesis; synergistic ⇒ bactericidal.

Consolidated Map (Target → Example Drug)

• Cell wall: β-lactams, vancomycin.
• Cell membrane: polymyxins, daptomycin.
• DNA gyrase/topo IV: fluoroquinolones.
• RNA polymerase: rifampin.
• Ribosome 30S: tetracyclines, aminoglycosides.
• Ribosome 50S: macrolides, clindamycin, chloramphenicol, linezolid.
• Folate pathway: sulfonamides, trimethoprim.

Antiviral Chemotherapy

• Viruses are obligate intracellular – must hijack host replication machinery.
• Key replication stages & drug blocks:

1. Attachment / fusion – enfuvirtide (HIV), docosanol (HSV), palivizumab (RSV).

2. Uncoating – amantadine, rimantadine (influenza).

3. Nucleic-acid synthesis – \text{NRTI/NNRTI} (HIV, HBV), aciclovir (HSV), foscarnet (CMV).

4. Integration – INSTIs (HIV).

5. Protein processing – protease inhibitors (HIV, HCV).

6. Viral release – neuraminidase inhibitors (oseltamivir for influenza).
   • HAART: ≥3 antiretrovirals from ≥2 classes ↓ viral load & resistance emergence.

Safety Issues with Antimicrobials (overview)

• Hypersensitivity (e.g. β-lactam allergy), nephro-/ototoxicity (aminoglycosides), QT prolongation (macrolides, fluoroquinolones), C. difficile colitis (broad-spectrum use).

Antimicrobial Resistance (AMR)

• Current deaths ≈ 7\times10^{5} year⁻¹; projected 2050 ≈ 1\times10^{7} year⁻¹ (exceeding cancer).
• WHO Priority Pathogens (2017):
– Critical: carbapenem-resistant Acinetobacter baumannii, Pseudomonas aeruginosa, ESBL-producing Enterobacteriaceae.
– High: VRE, MRSA/VRSA, fluoroquinolone-resistant Campylobacter, Salmonella, etc.
– Medium: penicillin-non-susceptible S. pneumoniae, ampicillin-resistant H. influenzae …

Types of Resistance

• Inherent (intrinsic): lack transport, lack target, outer-membrane barrier (Gram – vs vancomycin).
• Acquired: mutation or horizontal gene transfer (conjugation, transformation, transduction).
• Multi-drug resistance (MDR) e.g. P. aeruginosa; cross-resistance within same class (β-lactams).

Molecular Mechanisms (illustrated)

• Drug-inactivating enzymes: β-lactamases, aminoglycoside-modifying enzymes.
• Altered targets: PBP2a via mecA in MRSA; D-Ala→D-Lac in VRE; gyrA mutations vs quinolones.
• Efflux pumps: AcrAB-TolC (Gram –), Mef(A) (macrolide in streptococci).
• Reduced uptake: porin loss (OprD in P. aeruginosa vs carbapenems).
• Bypass pathways: ↑ PABA production or exogenous folate uptake to evade sulfonamides.

Detailed β-Lactam Resistance Example

• β-lactamase hydrolyses β-lactam ring \rightarrow penicilloic acid (inactive).
• ESBL genes on plasmids ⇒ rapid intra/inter-species spread.
• Altered PBPs: MRSA expresses PBP2a (low β-lactam affinity) encoded by mecA regulatory island.

Vancomycin Resistance

• Enterococci replace D-Ala-D-Ala with D-Ala-D-Lac terminal in PG precursor (VanA/VanB operon) ⇒ >1000-fold ↓ affinity.

Drivers of the Crisis

1. Overuse/misuse in humans – viral URTIs treated with antibiotics; example: children (0–6 yrs) in south Sweden average 13 days antibiotics/year.

2. Veterinary/agricultural – growth-promotion & mass prophylaxis (now banned in EU) created resistant Campylobacter, Salmonella transferring to humans.

Global & National Responses

• UK AMR Strategy 2013-2018: stewardship (responsible prescribing), infection-control, public awareness, research, new drugs/vaccines, surveillance, international collaboration.
• Research foci: epidemiology of spread, rapid diagnostics (bed-side genomics), novel targets, alternative therapies (bacteriophages, antibodies, probiotics, anti-virulence agents, immunomodulators).

Alternative / Adjunct Therapies (examples)

• Phage therapy – specific bacteriophages lyse MDR bacteria; challenge = narrow spectrum, regulatory hurdles.
• Monoclonal antibodies – neutralise toxins (e.g. bezlotoxumab vs C. difficile toxin B).
• Anti-virulence drugs – quorum-sensing inhibitors; disarm pathogens without killing ⇒ lower selection pressure.
• Probiotics & microbiome transplantation – restore colonisation resistance.
• CRISPR-Cas antimicrobials – gene editing to remove resistance plasmids.

Integrated View: Targets vs Resistance Strategies

• Cell wall ↔ β-lactamase, altered PBPs, van gene cluster.
• Membrane ↔ mcr-1-encoded phosphoethanolamine transferase (colistin resistance).
• DNA/RNA ↔ gyrA/topoIV mutations, rpoB mutation (rifampin).
• Protein synthesis ↔ ribosomal methylases (erm), 16S rRNA point mutations.
• Efflux pumps provide broad cross-class defence (fluoroquinolones, tetracyclines, macrolides).

"Post-Antibiotic Era" Warning

• Dr Margaret Chan (ex-WHO DG): everyday infections (strep throat, minor wounds) could again be lethal; jeopardises modern medicine (transplants, chemo, C-sections).

Take-Home Messages

• Understanding microbial targets & drug mechanisms aids rational therapy & stewardship.
• AMR arises via diverse genetic/biochemical strategies; human behaviour accelerates it.
• Integrated global action—new antibiotics, diagnostics, stewardship, vaccines & alternative modalities—is essential to avert projected 10 million AMR deaths/year by 2050.

Self-Check / Practice Questions

• Define MIC & MBC and explain how they are experimentally determined.
• Compare mechanism of action of β-lactams vs glycopeptides.
• Why are fluoroquinolones concentration-dependent killers whereas β-lactams are time-dependent?
• Outline four molecular mechanisms by which bacteria acquire resistance to aminoglycosides.
• Describe how HAART prevents HIV replication & discuss resistance management.
• Propose two alternative non-antibiotic therapies and evaluate their pros/cons.