Pharmacology of Antibiotics

Spectrum of Activity

• Single chemotherapeutic agent does not affect all microbes
• Spectrum of activity: types of organisms the agents can affect.
• Antibacterial, antiprotozoal, antiviral
• Antibacterial agents are further narrowed down based on the type of bacteria they affect:
  • Narrow spectrum: Penicillin --> Gram (+)
  • Broad spectrum: Ampicillin --> Gram (+) + Gram (-)
  • Very narrow spectrum: Isoniazid --> Mycobacterium tuberculosis
  • Ampicillin has an added amino group that helps it penetrate the gram-negative outer membrane

Bacteriostatic and Bactericidal Antibiotics

• Bacteriostatic antibiotics prevent bacterial growth, allowing the immune system to kill the pathogen.
• Bactericidal antibiotics kill the pathogens

Minimum Inhibitory Concentration (MIC)

• The lowest concentration of the drug that prevents the growth of an organism.
• Depends on the bacterial species, drug penetration ease, and drug affinity to its target.
• Measured by serially diluting an antibiotic in nutrient broth and inoculating with bacteria.
• The tube with the lowest concentration of the drug that shows no growth represents the MIC.
• MIC does not indicate whether the drug is bacteriostatic or bactericidal.
• Useful for estimating a single drug’s effectiveness against a bacterial pathogen.
• Strip test is a faster method that avoids dilutions.
• An elliptical zone of inhibition forms on an agar plate, with the MIC represented by the point of intersection between the zone and the strip.

Disk Diffusion Assay

• Kirby-Bauer assay is a standardized method that uses filter paper disks impregnated with different antibiotics.
• Disks are placed on an agar plate covered with a bacterial lawn.
• Drugs diffuse away from the disks, inhibiting bacterial growth at various distances.
• Zone of inhibition is measured around the disk, its size depends on the antibiotic, drug concentration, and bacterial susceptibility.
• Diameter of the zone correlates with the MIC.
• Standardized parameters ensure accurate results (agar plate size, medium depth, medium composition, organism number, disk size, antibiotic concentration, incubation temperature).
• Does not identify bactericidal or bacteriostatic action.

Minimal Bactericidal Concentration (MBC)

• Requires further plating to determine if cells survived after antibiotic treatment.
• Determines if the antibiotic kills the bacteria or only inhibits growth.
• Lowest concentration of the drug that does not show growth represents the MBC.
• Any antibiotic can be bactericidal at high concentrations.
• A drug is considered bactericidal if its MBC is no more than four times its MIC.

Clinical Considerations of Antibiotic Usefulness

• The concentration of the drug in tissue must exceed the MIC at all times.
• Half-life determines how long the drug remains in the tissue.
• High doses or multiple doses might be needed to maintain drug levels above the MIC.
• Antibiotic effectiveness depends on whether side effects occur at concentrations needed to affect the pathogen.
• Higher doses can lead to more side effects.
• Repeated doses at intervals help to ensure therapeutic levels.

Chemotherapeutic Index

• Therapeutic dose: Minimum dose per kg of body weight that stops pathogen growth.
• Toxic dose: Maximum dose tolerated by the patient.
• Chemotherapeutic index is the ratio of toxic dose to therapeutic dose.
• Higher index indicates a safer drug.
• Ideally, the therapeutic dose is much lower than the toxic dose.

Synergism and Antagonism in Antibiotic Combinations

• Synergistic drugs have greater effectiveness when used together (combined effect is greater than additive).
• Example: Aminoglycoside + vancomycin for serious enterococcus infections.
• Antagonistic drugs interfere with each other, decreasing effectiveness.
• Occurs when bacteriostatic and bactericidal agents are combined.
• Example: Penicillin + macrolides.

Antimicrobial Mechanisms of Action

• Antibacterial agents are classified based on:
  • Bactericidal or bacteriostatic action.
  • Target site.
  • Chemical structure.

Antibiotics that Target the Cell Wall:

• Inhibit the synthesis of peptidoglycan, a major component of bacterial cell walls.
• Peptidoglycan is composed of NAG and NAM, which are linked together.
• These antibiotics disrupt the synthesis and assembly of peptidoglycan, weakening the cell wall and leading to cell lysis.

Antibiotics that Target DNA Gyrase:

• DNA gyrase is a bacterial enzyme that relieves supercoiling in DNA during replication.
• Quinolones inhibit DNA gyrase, leading to accumulation of positive supercoils, inhibiting DNA replication.
• Bacterial DNA gyrase is different from mammalian DNA gyrase, making this mechanism selective.
• Examples: Ciprofloxacin and levofloxacin.

Antibiotics that Target RNA Synthesis:

• Rifampin inhibits bacterial RNA polymerase.
• Selectively binds to bacterial RNA polymerase, preventing newly made mRNA from exiting the enzyme, stopping transcription.
• Used to treat tuberculosis and meningococcal meningitis.

Antibiotics that Target Protein Synthesis:

• Molecular complexity of translation offers many sites for antibiotic attack.
• Target bacterial ribosomes, which differ from eukaryotic ribosomes.
• Most are bacteriostatic.
• Bind to and interfere with the function of bacterial rRNA.

Metronidazole:

• Prodrug that is harmless until activated by reduction.
• Activated by microbial protein cofactors (ferredoxin) found in anaerobic or microaerophilic bacteria.
• Human cells lack these cofactors, preventing activation in humans.
• Activated metronidazole damages DNA, killing anaerobic bacterial cells.
• Used to treat infections caused by anaerobic bacteria like Bacteroides and Fusobacterium.

Rifampin:

• A member of the rifamycin family.
• Selectively binds to bacterial RNA polymerase, preventing newly made mRNA from exiting the enzyme.
• Inhibits transcription.
• Used to treat tuberculosis and meningococcal meningitis.
• Turns urine and breast milk orange.

Microbial Resistance to Cell Wall-Inhibiting Antibiotics

• Bacteria develop resistance to penicillin in two ways:
  • Inheriting a gene encoding beta-lactamase enzymes: These enzymes cleave the beta-lactam ring structure of penicillin, rendering it ineffective. This enzyme is transported out of the cell and into the surrounding medium (Gram-positive) or the periplasm (Gram-negative) where it destroys penicillin before it reaches the cell.
  • Altering a key penicillin-binding protein by mutation: This altered protein can still synthesize peptidoglycan but will no longer bind penicillin. Most methicillin-resistant bacteria (MRSA) employ this strategy. Vancomycin can be used against MRSA.

Drugs that Target the Bacterial Membrane

• Gramicidin:
  • Produced by Brevibacillus brevis.
  • Cyclic peptide composed of 15 alternating D and L amino acids.
  • Inserts into the membrane as dimers, forming a cation channel that allows positive ions to freely cross the membrane, disrupting membrane polarity.
• Polymyxins:
  • Polymyxin B acts as a detergent.
  • It has a positively charged polypeptide ring that binds to the negatively charged outer and inner membranes of bacteria.
  • It dissolves the inner membrane by disrupting phospholipid interactions.
  • Used topically to treat or prevent infection.
  • Not ingested due to its ability to damage human cell membranes.
  • Used in some bandages for burn patients susceptible to Gram-negative bacteria (P. aeruginosa).

Drugs that Affect DNA Synthesis and Integrity

• Penicillin-Binding Proteins (PBPs):
  • Enzymes that attach disaccharide units to pre-existing peptidoglycan and produce peptide cross-links.
  • Penicillin and related antibiotics bind to these proteins.
  • Penicillin's structure resembles a piece of the peptidoglycan peptide side chain, allowing it to bind to PBPs and inhibit their activity.
• Impact of Penicillin on Cell Wall:
  • Penicillin prevents peptide cross-linking, weakening the bacterial cell wall.
  • Growing cells eventually burst from internal pressure due to the compromised cell wall.
  • Penicillin is a bactericidal drug.
  • Penicillin is more effective against Gram-positive bacteria as their outer membrane blocks penicillin entry in Gram-negative bacteria.

The Chemotherapeutic Index

• Therapeutic Dose: Minimum dose per kg of body weight that stops the growth of pathogens.
• Toxic Dose: Maximum dose tolerated by the patient.
• Chemotherapeutic Index: Ratio of the toxic dose to the therapeutic dose.
  • A higher index indicates a safer drug.
  • Ideally, the therapeutic dose is much lower than the toxic dose.

Synergism and Antagonism

• Synergistic Drugs: More effective when used together, resulting in an effect greater than the sum of their individual effects.
  • Example: Aminoglycoside (inhibits protein synthesis) & Vancomycin (cell wall synthesis inhibitor) used for serious enterococcus infections.
• Antagonistic Drugs: Interfere with each other, decreasing effectiveness.
  • Example: Penicillin (bactericidal) + Macrolides (bacteriostatic).
    • Penicillin kills actively growing cells synthesizing peptidoglycan, having little effect on non-growing cells.
    • Macrolides prevent bacterial growth.

Antimicrobial Mechanisms of Action: Classification

• Antibacterial agents can be classified based on:
  • Bactericidal or Bacteriostatic: Whether they kill bacteria or simply inhibit their growth.
  • Target Site: The specific cellular component targeted by the antibiotic.
  • Chemical Structure: The antibiotic's chemical makeup.

Antibiotics that Target the Cell Wall

• NAG and NAM: Short disaccharides made by the cell and linked together to form long peptidoglycan chains, which are assembled at the cell wall.

Explore the spectrum of activity of different antibiotics, distinguishing between bacteriostatic and bactericidal properties. Understand the concept of Minimum Inhibitory Concentration (MIC) and how it varies among bacterial species. This quiz covers key pharmacological principles related to antibiotic effectiveness.