ANTIBIOTICS

OUTLINE

a. Bacterial cell structure

b. Antibiotic groups and sites of action

c. Mechanisms of Resistance in Bacteria

d. Sensitivity testing

Principles and Definitions• Selectivity

– Selectivetoxicity• Therapeutic index

– Toxic dose/ Effective dose• Categories of antibiotics

– Bactericidal• Usually antibiotic of choice

– Bacteriostatic• Duration of treatment sufficient for host defenses

Principles and Definitions

• Antibiotic susceptibility testing (in vitro)– Minimum inhibitory concentration (MIC)

• Lowest concentration that results in inhibition of visible growth

– Minimum bactericidal concentration (MBC)• Lowest concentration that kills 99.9% of the original

inoculum

Inhibitors of cell wall synthesis

A Beta lactam antibiotics Penicillins Cephalosporins B Glycopeptides C Bacitracin spheroplasts and protoplasts

CytoplasmCytoplasm

Lipoteichoic acid Peptidoglycan-teichoic acid

Cytoplasmic membrane

GRAM POSITIVE CELL GRAM POSITIVE CELL ENVELOPEENVELOPE

Degradative enzyme

GRAM NEGATIVE GRAM NEGATIVE CELL ENVELOPECELL ENVELOPE

CytoplasmCytoplasm

Inner (cytoplasmic) membrane

Outer Membrane(Major permeability barrier) LipopolysaccharidePorin

Braun lipoprotein

Periplasmic space Degradative enzyme

Periplasmic binding protein Permease

Inhibitors of Cell Membrane function

1. Detergents: Colistin and Polymyxins2. Inhibition of Biosynthetic Functions:

Nalidixic acid and Novobiocin Polyenes3. Ionophores: eg K+ loss

Antifungals

Inhibitors of protein synthesis 70S versus 80S ribosomes

30S

AMINOGLYCOSIDES

TETRACYCLINES

OXAZOLIDINONES

50S

CHLORAMPHENICOL

MACROLIDES

LINCOMYCIN

Protein Synthesis Inhibitors

• Mostly bacteriostatic• Selectivity due to differences in

prokaryotic and eukaryotic ribosomes• Some toxicity -

• Aminoglycosides 1 receptor attachment 2 prevents formation of initiation complex 3 misreading 4 polysomes break into monosomes

Inhibition of Nucleic Acid Synthesis

DNA• Quinolones DNA gyrase

• Sulfonamides• Trimethoprim RNA• Rifampicin RNA polymerase

Inhibitors of RNA Synthesis

Selectivity due to differences between

prokaryotic and eukaryotic RNA polymerase

Inhibitors of DNA Synthesis

Selectivity due to differences between

prokaryotic and eukaryotic enzymes

Quinolones (bactericidal)nalidixic acid, ciprofloxacin, ofloxacin, norfloxacin,

levofloxacin, lomefloxacin, sparfloxacin

• Mode of action - These antimicrobials bind to the A subunit of DNA gyrase (topoisomerase) and prevent supercoiling of DNA, thereby inhibiting DNA synthesis.

• Spectrum of activity - Gram-positive cocci and urinary tract infections

• Resistance - Common for nalidixic acid; developing for ciprofloxacin

Rifampin, Rifamycin, Rifampicin, Rifabutin (bactericidal)

• Mode of action - These antimicrobials bind to DNA-dependent RNA polymerase and inhibit initiation of mRNA synthesis.

• Spectrum of activity - Broad spectrum but is used most commonly in the treatment of tuberculosis

• Resistance - Common

• Combination therapy - Since resistance is common, rifampin is usually used in combination therapy.

Inhibitors of Folic Acid Synthesis

• Basis of Selectivity

• Review of Folic Acid Metabolism

PABA+ Pteridine

Dihydropteroic acid

Dihydrofolic acid

Tetrahydrofolic acid

Pteridine synthetase

Dihydrofolate synthetase

Dihydrofolate reductase

Purines

Trimethoprim

Sulfonamide

• Most antimicrobials have more than one mechanism of action.

Mechanisms of Antimicrobial Resistance

Beta-lactam Antibiotics

Mechanism of Action ofBeta-lactams

Penicilllin, action of -lactamase

Site of amidase action

Site of penicillinase action(break in-lactam ring)

Beta-lactamase resistantBeta-lactams

Mechanism of Methicillin Resistance

SensitivityTesting

Antibiotic susceptibility testing (in vitro)

Minimum inhibitory concentration (MIC)Lowest concentration that results in inhibition of visible growth

Minimum bactericidal concentration (MBC)Lowest concentration that kills 99.9% of the original inoculum

Antibiotic Susceptibility Testing

8 4 02 1 Tetracycline (g/ml)

MIC = 2 g/ml

Determination of MIC

Chl Amp

Ery

Str

Tet

Disk Diffusion Test

Media used for Sensitivity Testing

• Mueller Hinton Agar

• DST

• Special media e.g. Chocolate agar

Disc Diffusion Testing

• A. Kirby Bauer method…… CLSI recommended

• B. Stokes Comparitive method

Kirby Bauer Test

MIC evaluation by E test

• Inoculum Standardization 0.5 McFarland Standard

Review

• Sites of action• Mechanisms of action• Major groups• Sensitivity testing methodology