Antimicrobial Chemotherapy & Resistance – Core Vocabulary

Learning Outcomes

The lecture sets five overarching competencies that students should achieve:

1. Describe how innate and adaptive immune defences interact with microorganisms, the pathological consequences of infection and the principles behind immunisation or immunotherapy.

2. Perform, troubleshoot and interpret laboratory procedures for isolating and diagnosing specific pathogens.

3. Appreciate the normal (commensal) microbiota, recognise the wide taxonomic range of pathogenic microbes and select suitable diagnostic strategies.

4. Discuss, at molecular, cellular and host levels, the principles of antimicrobial chemotherapy and the problems posed by resistance.

5. Explain common pathogenic determinants/processes and outline the pathogenesis of named diseases.

Overview of Antimicrobial Chemotherapy

Antimicrobial chemotherapy is the use of drugs to combat infectious agents. Four broad categories exist:

• Antibacterial

• Antifungal

• Antiparasitic

• Antiviral

The key pharmacological concept is differential toxicity – the agent must be more toxic to the pathogen than to the human host.

Types of Antimicrobial Agents

1. Antibacterial agents – largest, most diverse group.

2. Antifungals – target ergosterol or fungal‐specific pathways.

3. Antiparasitics – protozoa and helminths; usually highly specific.

4. Antivirals – must work inside host cells and target virus‐specific steps.

Discovery and Development of Antibiotics

• 1900 – Salvarsan (Paul Ehrlich) introduced for syphilis.

• 1928 – Penicillin discovered accidentally by Alexander Fleming (Penicillium notatum).

• 1942 – Large-scale US production (WWII) begins, saving millions of lives.

• Pre-antibiotic era relied on cytotoxic metals: silver nitrate, arsenicals.

Recent pipeline example: Teixobactin – first-in-class cell-wall inhibitor expected within the next ≈5 years.

Socio-economic advances—improved surgery, transplantation, intensive care—are inseparable from effective antibiotics.

Antibiotic-Producing Microorganisms (Natural Sources)

• Gram-positive rods
– Bacillus subtilis → Bacitracin
– Bacillus polymyxa → Polymyxin

• Fungi
– Penicillium notatum → Penicillin
– Cephalosporium spp. → Cephalothin

• Actinomycetes
– Streptomyces venezuelae → Chloramphenicol
– Streptomyces griseus → Streptomycin
– Streptomyces nodosus → Amphotericin B
– Micromonospora purpurea → Gentamicin

Characteristics of an Ideal Antibiotic

• Selective toxicity; minimal collateral damage.

• Cidal activity (kills) rather than merely static (inhibits).

• Long biological half-life; infrequent dosing.

• Good tissue penetration (including CSF if required).

• Oral and parenteral formulations.

• Low rate of resistance emergence.

• Minimal drug–drug interactions and adverse events.

• Cost-effective and chemically stable.

Spectrum of Activity

• Narrow-spectrum – effective against a limited group (e.g. Gram-positives only). Example: Penicillin G, \text{Isoniazid} (Mycobacterium-specific).

• Broad-spectrum – effective against diverse Gram-positive and Gram-negative species. Example: Tetracycline.

Clinicians often begin with a broad agent empirically, then “de-escalate” to a narrow agent once organism is known.

Key Quantitative Definitions

• Minimum Inhibitory Concentration (MIC): \text{lowest [drug]} that visibly inhibits growth.

• Minimum Bactericidal Concentration (MBC): \text{lowest [drug]} that kills ≥99.9\% of the inoculum.

• Bacteriostatic – reversibly inhibits replication.

• Bactericidal – kills outright.

• Time-dependent killing – efficacy linked to \text{T}>\text{MIC} (e.g. \beta-lactams).

• Concentration-dependent killing – efficacy linked to \text{C}_{\text{max}}/\text{MIC} or \text{AUC}/\text{MIC} (e.g. aminoglycosides).

Prophylaxis vs Treatment

• Prophylaxis – pre-emptive antimicrobial administration to prevent infection (e.g. surgical prophylaxis, malaria prophylaxis).

• Treatment – therapeutic administration to eradicate an established/suspected infection.

Bacterial Cell Architecture – Why It Matters

1. Cell wall (peptidoglycan) – provides shape, osmotic protection; absent in humans → prime drug target.

2. Cell (plasma) membrane – phospholipid bilayer regulating influx/efflux.

3. Outer membrane (Gram-negative only) – lipopolysaccharide (LPS) barrier, porins.

4. Ribosomes (70S) – distinct from eukaryotic 80S, enabling selective inhibition of translation.

5. Nucleoid DNA – circular dsDNA plus plasmids; differences in gyrase/topoisomerase/RNA polymerase exploited by drugs.

Gram-Positive vs Gram-Negative – Structural & Staining Differences

• Gram-positive: thick peptidoglycan with teichoic/lipoteichoic acids; retains crystal violet–iodine complex → purple.

• Gram-negative: thin peptidoglycan, outer LPS membrane; crystal violet washed out, safranin counterstain → pink/red.

Gram staining steps: Crystal violet → Gram’s iodine (mordant) → 95\% ethanol de‐colourisation (<30 s) → Safranin.

Major Antibacterial Target Sites & Representative Classes

1. Inhibition of cell wall synthesis – \beta-lactams, glycopeptides.

2. Disruption of cell membrane – polymyxins, daptomycin.

3. Inhibition of nucleic acid synthesis – quinolones, rifamycins, metronidazole.

4. Inhibition of protein synthesis – macrolides, tetracyclines, aminoglycosides, chloramphenicol, lincosamides, streptogramins.

5. Antimetabolites (folate pathway) – sulfonamides, trimethoprim.

1 – Inhibition of Cell-Wall Synthesis

\beta-Lactams

Mechanism: bind Penicillin-Binding Proteins (PBPs) – transpeptidases that cross-link peptidoglycan. Binding blocks cross-linking → weakened wall → osmotic lysis. Time-dependent, bactericidal.

Sub-classes:

• Penicillins

• Cephalosporins

• Cephamycins

• Carbapenems (broadest)

• Monobactams (not covered in slides but clinically relevant)

Glycopeptides (Vancomycin, Teicoplanin)

Mechanism: bind terminal D\text{-}Ala–D\text{-}Ala dipeptide of nascent cell-wall units → steric blockade of PBPs; prevents cross-linking. Bactericidal against Gram-positives.

2 – Disruption of Cell Membrane

Polymyxin B, Colistin (polymyxin E) – cationic peptides attracted to negatively charged LPS of Gram-negatives. Insert into membrane → pore formation → leakage of ions/metabolites → rapid death. Time-dependent.

Antifungal parallel: Miconazole targets ergosterol, increasing fungal membrane permeability.

3 – Inhibition of Nucleic Acid Synthesis

Quinolones / Fluoroquinolones (ciprofloxacin, levofloxacin, moxifloxacin)

Bind two essential topoisomerases:

• DNA gyrase (Gram-negatives)

• Topoisomerase IV (Gram-positives)
  → DNA double-strand breaks during replication. Concentration-dependent, bactericidal.

Rifamycins (rifampicin, rifabutin)

High affinity for bacterial RNA polymerase β-subunit → block DNA-dependent RNA synthesis. Key drugs for Mycobacterium tuberculosis, M. leprae; useful synergy against Gram-positives.

4 – Inhibition of Protein Synthesis

Bacterial ribosomes are 70S (50S + 30S). Multiple classes act at distinct sites:

• Macrolides (erythromycin, azithromycin) – bind 50S exit tunnel; bacteriostatic, time-dependent, excellent for atypicals (Mycoplasma, Chlamydia).

• Ketolides – semi-synthetic macrolide derivatives, improved PBP affinity.

• Lincosamides (clindamycin) – 50S inhibitor, active vs Gram-positives & anaerobes.

• Streptogramins – synergistic 50S binders (quinupristin/dalfopristin).

• Tetracyclines – block aminoacyl-tRNA docking on 30S; broad spectrum.

• Aminoglycosides (gentamicin, tobramycin, amikacin) – cause mRNA mis-reading at 30S; concentration-dependent, bactericidal, strong Gram-negative activity.

• Chloramphenicol – inhibits peptidyl transferase on 50S; broad spectrum but serious toxicity (aplastic anaemia).

Illustrative binding sites: streptomycin distorts 30S decoding centre; tetracyclines block tRNA entry; chloramphenicol blocks peptide-bond formation; macrolides block elongation tunnel.

5 – Antimetabolites (Folate Pathway)

Bacteria must synthesise tetrahydrofolate (THF) de novo to produce purines and some amino acids.

• Sulfonamides (SMX) – competitive analogues of p\text{-}aminobenzoic\;acid (PABA); inhibit dihydropteroate synthase.

• Trimethoprim (TMP) – inhibits dihydrofolate reductase.

Combined as \text{TMP} + \text{SMX} (cotrimoxazole) → sequential blockade, synergistic, bactericidal. Key for Gram-negative UTIs, some Gram-positives (S. pneumoniae).

Antiviral Chemotherapy

Viruses are obligatory intracellular parasites; drug targets must be virus-specific yet spare host.

Key viral life-cycle stages and corresponding drug classes:

1. Attachment / Entry – Enfuvirtide (HIV), Docosanol (HSV), Palivizumab (RSV).

2. Uncoating – Amantadine, Rimantadine (influenza A).

3. Nucleic-acid synthesis – Nucleoside RT inhibitors (NRTIs) & Non-nucleoside RT inhibitors (NNRTIs) for HIV; Acyclovir (HSV), Foscarnet (CMV).

4. Integration – Integrase strand-transfer inhibitors (INSTIs) for HIV.

5. Protein processing – Protease inhibitors (PIs) for HIV/HCV.

6. Virion release – Neuraminidase inhibitors (oseltamivir, zanamivir) for influenza.

HAART (Highly Active Antiretroviral Therapy)

Combination of ≥3 drugs acting on ≥2 distinct targets dramatically lowers HIV viral load.

• Reverse-Transcriptase Inhibitors (RTIs)

• Protease Inhibitors (PIs)

• Fusion/Entry/CCR5 inhibitors

• Integrase inhibitors

Safety Issues with Antimicrobials

Adverse effects range from mild GI upset to life-threatening anaphylaxis, nephrotoxicity (aminoglycosides), ototoxicity, bone marrow suppression (chloramphenicol) and Clostridioides difficile colitis (broad-spectrum agents).

Antimicrobial Resistance (AMR)

Defined as the ability of a microbe to withstand levels of drug that would normally inhibit/kill. Already causes ≈700\,000 deaths annually; projected 10 million deaths/year by 2050 (Review on AMR, 2015).

WHO Priority Pathogens (2017):

• Priority 1 – Critical: A.\;baumannii (carbapenem-R), P. aeruginosa (carbapenem-R), ESBL-producing Enterobacteriaceae.

• Priority 2 – High: VRE, MRSA/VRSA, clarithromycin-R H. pylori, fluoroquinolone-R Campylobacter/Salmonella, cephalosporin- / fluoroquinolone-R N. gonorrhoeae.

• Priority 3 – Medium: Penicillin-NS S. pneumoniae, ampicillin-R H. influenzae, fluoroquinolone-R Shigella.

Types of Resistance

1. Inherent (intrinsic) – natural lack of drug target, impermeable outer membrane, or efflux pumps (e.g. P. aeruginosa).

2. Acquired – mutation or horizontal gene transfer (conjugation, transformation, transduction).

3. Multi-resistance – resistance to multiple classes (e.g. MRSA, MDR P. aeruginosa).

4. Cross-resistance – within same chemical class (all \beta-lactams).

Molecular Mechanisms (illustrated slide)

• Enzymatic drug inactivation – \beta-lactamases, aminoglycoside-modifying enzymes.

• Reduced uptake – porin loss/mutation.

• Active efflux – multidrug transporters.

• Target modification – altered PBPs (mecA → PBP2a in MRSA), mutated gyrA/parC (quinolones), methylated 23S rRNA (macrolide resistance).

• Bypass pathways – acquisition of alternate enzymes (VanA → D-Ala–D-Lac terminal, confers vancomycin resistance).

Beta-Lactam Resistance Examples

1. \beta-lactamases (penicillinases, cephalosporinases, carbapenemases, ESBLs) hydrolyse the \beta-lactam ring → inactive penicilloic acid.

2. Altered PBPs – mecA gene in MRSA encodes PBP2a with low affinity for \beta-lactams.

Vancomycin Resistance (VRSA/VRE)

Replacement of terminal D\text{-}Ala with D\text{-}Lac in peptidoglycan precursors → 1000-fold reduced vancomycin affinity.

Causes of the Current AMR Crisis

1. Medical overuse – prescribing for viral URTIs; Swedish data: average child (0-6 y) receives 13 days of antibiotics per year.

2. Veterinary/agricultural use – growth promotion & mass prophylaxis foster resistant zoonotic pathogens (Salmonella, Campylobacter). EU has banned growth-promotion use.

Combating Resistance – UK 2013-18 Strategy

1. Promote responsible prescribing (antibiotic stewardship).

2. Strengthen infection-prevention & control (IPC) in human/animal sectors.

3. Public & professional awareness campaigns.

4. Invest in basic & translational research.

5. Develop new drugs, vaccines, diagnostics.

6. Enhance national & international surveillance.

7. Foster global collaboration.

Research Questions Going Forward

1. Epidemiology – mapping dissemination routes of resistant clones/genes.

2. Diagnostics – rapid bedside genomics/phenotypic assays to guide therapy.

3. Novel Targets – essential, non-redundant bacterial proteins/metabolic pathways.

4. Alternative Therapies – bacteriophages, anti-virulence agents, probiotics, CRISPR-based editing, immunomodulators.

Alternative Therapies – Pros & Cons (Prompted for Discussion)

• Phage therapy – highly specific, self-replicating; regulatory & immune concerns.

• Bacteriocins / antimicrobial peptides – potent, but stability & toxicity issues.

• Monoclonal antibodies – target toxins; expensive.

• Fecal microbiota transplantation – effective for recurrent C. difficile; logistics & safety considerations.

• Quorum-sensing inhibitors – disarm pathogens without killing; less selective pressure but still under development.

Consolidated Map of Targets vs Resistance Mechanisms (Slide 71)

• Cell wall – \beta-lactams, vancomycin vs \beta-lactamases, altered PBPs, VanA/B.
• Cell membrane – daptomycin vs membrane charge alteration.
• Protein synthesis – tetracyclines, macrolides, linezolid vs efflux, methylases, rRNA mutations.
• DNA/RNA synthesis – fluoroquinolones, rifamycins vs gyrase/topo mutations, efflux, RNA-pol mutations.
• Folate synthesis – sulfonamides, trimethoprim vs target‐site mutations or bypass.

Closing Perspective

WHO’s former Director-General Dr Margaret Chan warned: “Things as common as strep throat or a child’s scratched knee could once again kill… a post-antibiotic era means, in effect, an end to modern medicine as we know it.” The imperative to preserve current drugs, develop new interventions and deploy diagnostics wisely is therefore both medical and societal.

Practice Question (Self-Test)

Describe at least three distinct molecular mechanisms by which bacteria can acquire resistance to \beta-lactam antibiotics, and outline one clinical strategy to overcome each mechanism.