Antimicrobial agents

Antibiotics Molecular targets

Principles and Definitions• Selectivity

– Selectivityvstoxicity

• Therapeutic index– Toxic dose/ Effective dose

• Categories of antibiotics– Bacteriostatic

• Reversibly inhibit growth • Duration of treatment sufficient for host defenses to

eradicate infection

– Bactericidal-• Kill bacteria• Usually antibiotic of choice for infections in sites such as

endocardium or the meninges where host defenses are ineffective.

Principles and Definitions

• Selectivity

• Therapeutic index• Categories of antibiotics

– Use of bacteriostatic vs bactericidal antibiotic• Therapeutic index better for bacteriostatic

antibiotic

• Resistance to bactericidal antibiotic

• Protein toxin mediates disease – use bacteriostatic protein synthesis inhibitor to immediately block synthesis of toxin.

Principles and Definitions

• Antibiotic susceptibility testing (in vitro)– Bacteriostatic Antibiotics

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

(colonies on a plate or turbidity of liquid culture)

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

Antibiotic Susceptibility Testing-MIC

8 4 02 1 Tetracycline (mg/ml)

MIC = 2 mg/ml

Determination of MIC

Chl Amp

Ery

Str

Tet

Disk Diffusion Test

Size of zone of inhibition depends on sensitivity, solubility, rate of diffusion. Compare results to MIC tables generated using standards.

Zone diameter (mm) Approx. MIC(ug/ml) for:Antimicrobial agent

(amt. per disk)and organism R I MS S R S

Ampicillin (10ug/disk)

Enerobacteriacae <11 12-13 >14 >32 <8

Haemophilus spp. <19 >20 >4 <2

Enterococci <16 >17 >16

Tetracycline (30 g) <14 15-18 >19 >16 <4

Zone Diameter Standards for Disk Diffusion Tests

Principles and Definitions• Combination therapy

– Prevent emergence of resistant strains– Temporary treatment until diagnosis is made– Take advantage of antibiotic synergism

• Penicillins and aminoglycosides inhibit cell wall synthesis and allow aminoglycosides to enter the bacterium and inhibit protein synthesis.

• CAUTION: Antibiotic antagonism– Penicillins and bacteriostatic antibiotics. Cell wall synthesis

is not occurring in cells that are not growing.

• Antibiotics vs chemotherapeutic agents vs antimicrobials– Antibiotics-naturally occurring materials– Chemotherapeutic-synthesized in the lab (most

antibiotics are now synthesized and are therefore actually chemotherapeutic agents.

Antibiotics that Inhibit Protein Synthesis

•Inhibitors of INITATION

•30S Ribosomal Subunit (Aminoglycosides, Tetracyclines, Spectinomycin)

•50S Ribosomal Subunit (Chloramphenicol, Macrolides)

•Inhibitors of ELONGATION

•Elongation Factor G (Fusidic acid)

Initiation of Protein Synthesis

30S1 32 GTP

1 2 3 GTP

Initiation Factors

mRNA

3

1

2 GTP

30S Initiation Complex

f-met-tRNA

Spectinomycin

Aminoglycosides

12

GDP + Pi 50S

70S Initiation Complex

AP

Elongation of Protein Synthesis

GTP

AP

Tu GTP Tu GDP

Ts

TsTu

+

GDPTs

Pi

P ATetracycline

AP

Erythromycin

Fusidic Acid

Chloramphenicol

G GTPG GDP + Pi

G

GDP

AP

+GTP

Survey of Antibiotics

Discuss one prototype for each category:

•Mode of Action

•Spectrum of Activity

•Resistance

•Synergy or Adverse Effects

Protein Synthesis Inhibitors

• Mostly bacteriostatic

• Selectivity due to differences in prokaryotic and eukaryotic ribosomes

• Some toxicity - 70S ribosomes eukaryotic in mitochondria

Antimicrobials that Bind to the 30S Ribosomal Subunit

Aminoglycosides (only bactericidal protein synthesis

inhibitor)streptomycin, kanamycin, gentamicin, tobramycin,

amikacin, netilmicin, neomycin (topical)

• Modes of action - – Irreversibly bind to the 16S ribosomal RNA and freeze

the 30S initiation complex (30S-mRNA-tRNA) and prevents initiation of translation.

– Increase the affinity of the A site for t-RNA regardless of the anticodon specificity. Induces misreading of the mRNA for proteins already being synthesized.

– Destabilize microbial membranes– Multiple modes of action is the reason this protein

synthesis inhibitor is bactericidal.

Aminoglycosides (bactericidal)streptomycin, kanamycin, gentamicin, tobramycin,

amikacin, netilmicin, neomycin (topical)

• Spectrum of Activity -Many gram-negative and some gram-positive bacteria; Not useful for anaerobic (oxygen required for uptake of antibiotic) or intracellular bacteria.

• Resistance - Common• Synergy - The aminoglycosides synergize with beta-

lactam antibiotics. The beta - lactams inhibit cell wall synthesis and thereby increase the permeability of the membrane to aminoglycosides.

Tetracyclines (bacteriostatic)tetracycline, minocycline and doxycycline

• Mode of action - The tetracyclines reversibly bind to the 30S ribosome and inhibit binding of aminoacyl-t-RNA to the acceptor site on the 70S ribosome.

• Spectrum of activity - Broad spectrum; Useful against intracellular bacteria

• Resistance - Common

• Adverse effects - Destruction of normal intestinal flora resulting in increased secondary infections; staining and impairment of the structure of bone and teeth. Not used in children.

Spectinomycin (bacteriostatic)

• Mode of action - Spectinomycin reversibly interferes with m-RNA interaction with the 30S ribosome. It is structurally similar to the aminoglycosides but does not cause misreading of mRNA. Does not destabilize membranes, and is therefore bacteriostatic

• Spectrum of activity - Used in the treatment of penicillin-resistant Neisseria gonorrhoeae

• Resistance - Rare in Neisseria gonorrhoeae

Antimicrobials that Bind to the 50S Ribosomal Subunit

Chloramphenicol, Lincomycin, Clindamycin

(bacteriostatic)

• Mode of action - These antimicrobials bind to the 50S ribosome and inhibit peptidyl transferase activity. No new peptide bonds formed.

• Spectrum of activity - Chloramphenicol - Broad range;Lincomycin and clindamycin - Restricted

range

• Resistance - Common

• Adverse effects - Chloramphenicol is toxic (bone marrow suppression) but is used in life threatening situations such as the treatment of bacterial meningitis.

Macrolides (bacteriostatic)erythromycin, clarithromycin, azithromycin, spiramycin

• Mode of action - The macrolides inhibit translocation of the ribosome.

• Spectrum of activity - Gram-positive bacteria, Mycoplasma, Legionella

• Resistance - Common

Antimicrobials that Interfere with Elongation Factors

Selectivity due to differences in prokaryotic and eukaryotic elongation factors

Fusidic acid (bacteriostatic)

• Mode of action - Fusidic acid binds to elongation factor G (EF-G) and inhibits release of EF-GDP from the EF-G/GDP complex. Can’t reload EF-G with GTP.

• Spectrum of activity - Gram-positive cocci

Inhibitors of Nucleic Acid Synthesis

Inhibitors of RNA Synthesis

Selectivity due to differences between prokaryotic and eukaryotic

RNA polymerase

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. Develops rapidly (RNA polymerase mutations)

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

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 alpha 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

Antimetabolite Antimicrobials

Inhibitors of Folic Acid Synthesis

Tetrahydrofolate required for the methyl group on methionine, and for thymidine and purine synthesis.

p-aminobenzoic acid + Pteridine

Dihydropteroic acid

Dihydrofolic acid

Tetrahydrofolic acid

Pteridine synthetase

Dihydrofolate synthetase

Dihydrofolate reductase

ThymidinePurines

Methionine

Trimethoprim

Sulfonamides

Sulfonamides, Sulfones (bacteriostatic)

• Mode of action - These antimicrobials are analogues of para-aminobenzoic acid and competitively inhibit pteridine synthetase, block the formation of dihydropteroic acid.

• Spectrum of activity - Broad range activity against gram-positive and gram-negative bacteria; used primarily in urinary tract and Nocardia infections.

• Resistance - Common

• Combination therapy - The sulfonamides are used in combination with trimethoprim; this combination blocks two distinct steps in folic acid metabolism and prevents the emergence of resistant strains.

Trimethoprim, Methotrexate, Pyrimethamine (bacteriostatic)

• Mode of action - These antimicrobials binds to dihydrofolate reductase and inhibit formation of tetrahydrofolic acid.

• Spectrum of activity - Broad range activity against gram-positive and gram-negative bacteria; used primarily in urinary tract and Nocardia infections.

• Resistance - Common

• Combination therapy - These antimicrobials are used in combination with the sulfonamides; this combination blocks two distinct steps in folic acid metabolism and prevents the emergence of resistant strains.

Anti-Mycobacterial Antibiotics

Para-aminosalicylic acid (PSA) (bacteriostatic)

• Mode of action - Similar to sulfonamides- competitively inhibit pteridine synthetase, block the formation of dihydropteroic acid

• Spectrum of activity - Specific for Mycobacterium tuberculosis

Dapsone (bacteriostatic)

• Mode of action - Similar to sulfonamides- competitively inhibit pteridine synthetase, block the formation of dihydropteroic acid

• Spectrum of activity - Used in treatment of leprosy (Mycobacterium leprae)

Antimicrobial Drug ResistancePrinciples and Definitions

• Clinical resistance vs actual resistance• Resistance can arise by new mutation or by gene

transfer (e.g. acquisition of a plasmid)• Resistance provides a selective advantage.• Resistance can result from single or multiple steps• Cross resistance vs multiple resistance

– Cross resistance -- Single mechanism-- closely related antibiotics are rendered ineffective

– Multiple resistance -- Multiple mechanisms -- unrelated antibiotics. Acquire multiple plasmids. Big clinical problem.

Antimicrobial Drug ResistanceMechanisms

• Altered permeability– Altered influx

• Mutation in a transporter necessary to import antibiotic can lead to resistance.

– Altered efflux• Acquire transporter gene that will pump the antibiotic out

(Tetracycline)

Antimicrobial Drug ResistanceMechanisms

• Inactivation of the antibiotic-lactamase

Chloramphenicol Acetyl Transferase

Antimicrobial Drug ResistanceMechanisms

• Mutation in the target site.– Penicillin binding proteins (penicillins)– RNA polymerase (rifampin)– 30S ribosome (streptomycin)

Antimicrobial Drug ResistanceMechanisms

• Replacement of a sensitive enzyme with a resistant enzyme– Plasmid mediated acquisition of a resistant

enzyme (sulfonamides, trimethoprim)