Lectures 5-6: Antimicrobic Agents and Resistance

Antibiotic

Chemical substance that has the capacity to inhibit the growth of or kill bacterial cells

Bactericidal

Kills bacterial cells

Bacteriostatic

Inhibits growth

Do not use with immunocompromised patients

Selective toxicity

More harmful to the bacterial cell than to host cells

Five classes of antibiotics based on their mode of action

1.) Inhibit cell wall synthesis

2.) Disrupt cell membrane function

3.) Inhibit protein synthesis

4.) Inhibit nucleic acid synthesis

5.) Antimetabolites

Peptidoglycan synthesis

NAG and NAM subunits are synthesized in the bacterial cytoplasm

Amino acids are added to them as a side chain

NAG-NAM units are transported across the cell membrane into the periplasm

AA side chains from consecutive layers are cross-linked by the transpeptidase enzyme

Inhibitors of Cell Wall Synthesis

Bactericidal

Beta-lactam antibiotics

Binds to & blocks the activity of transpeptidases, preventing cross-linking of peptidoglycan layers

Absence of cross-links makes peptidoglycan disintegrate and bacteria burst due to loss of osmoregulation

Glycopeptides (vancomycin)

Gram-positives resistant to other antibiotics

Mode of action: Blocking the transglycosylation & transpeptidation steps of peptidoglycan synthesis

Bacitracin

Topical use only due to toxicity

Mode of action: Preventing assembly & transport of the NAG-NAM

Fosfomycin

Urinary tract infections

Mode of action: Preventing the formation of NAM (N-acetylmuramic acid)

Polymyxin

Topical or ophthalmic use only

Mode of action: Acts as a cationic detergent, disrupting membrane structure

Bactericidal

Daptomycin

Mode of action: disrupts cell membrane function by inserting and aggregating in the membrane, causing ion leakage that results in rapid membrane depolarization

Bactericidal

Aminoglycosides

Mode of action: Irreversibly bind to the 30S subunit to block the initiation complex

Bactericidal

Tetracyclines

Mode of action: Binds to the 30S subunit to prevent attachment of the aminoacyl-tRNA to the RNA-ribosome complex

Bacteriostatic

Contraindicated in children with developing teeth and pregnant women

Chloramphenicol

Poor selective toxicity; toxic to human mitochondria

Mode of action: Binds to the 50S subunit, inhibiting peptidyl transferase

Bacteriostatic

Macrolides

Allergic to penicillins

Mode of action: Binds to the 50S subunit, constricts the polypeptide exit tunnel, preventing chain elongation

Causes premature detachment 

Bacteriostatic

Lincosamides

Mode of action: Binds to the 50S subunit; has a similar mechanism of action as macrolides; disrupts protein synthesis

Bacteriostatic

Streptogramins

Mode of action: Bind to different sites on the 50S ribosomal subunit; inhibition of protein synthesis at different steps

Separately, streptogramins A and B are bacteriostatic; bactericidal together

Oxazolidinones

Mode of action: Binds to the P site of the 50S ribosomal subunit; prevents formation of the larger ribosomal complex

Bacteriostatic

Quinolones/Fluoroquinolones

Mode of action: Bind to and inhibit DNA gyrase (topoisomerase II) and topoisomerase IV

Bactericidal

Rifamycins

Mode of action: Inhibits DNA-dependent RNA polymerase

Bactericidal

Metronidazole

Mode of action: Covalently binds DNA, causing DNA breaks

Bactericidal

Sulfonamides & Trimethoprim

Mode of action: Inhibits folic acid synthesis (needed for DNA synthesis)

Bacteriostatic individually; synergistic in combination: Bactrim

Isoniazid

Mode of action: Is a prodrug that is activated by the bacterial enzyme KatG (catalase-peroxidase enzyme); active INH inhibits mycolic acid synthesis

Bactericidal against actively growing bacteria

Innate (intrinsic) resistance

Innate ability of a bacterial species to resist the activity of an antibiotic through its inherent structural or functional characteristics

Chromosomally encoded

Relates to general physiology arising from existing properties

Acquired (extrinsic) resistance

Organism obtains the ability to resist the activity of an antibiotic to which it was previously susceptible

Mutation in a resident gene

Transfer of genetic material encoding a resistance gene via vertical or horizontal gene transfer

Horizontal gene transfer

Transformation: Uptake of naked DNA from a donor cell to a recipient cell

Transduction: Phage-mediated transfer of bacterial DNA from a donor to a recipient

Conjugation: Transfer of plasmid DNA from a donor to a recipient cell during cell-to-cell contact

Bacterial Resistance

1.) Reduce the ability of the antibiotic to enter the cell- Altering porins in the cell wall

2.) Expel the antibiotic out of the cell via efflux pumps

3.) Antibiotic inactivation by modification or degradation:  Beta-lactamases

4.) Modification of the antimicrobial target

5.) Bypassing the antibiotic target: Some bacteria have acquired a “new” enzyme that allows bypass of a metabolic pathway

Beta-Lactamases Inhibitors

Used in combination with a beta-lactam antibiotic to protect it from beta-lactamase enzymes

Structural analogs of beta-lactams

Inhibit the activity of beta-lactamase

Disk diffusion

Surface of a Mueller-Hinton agar plate is evenly inoculated with a standardized suspension of the bacterial isolate to be tested

Filter paper disks containing antibiotics are aseptically placed onto the surface of the plate and the plate is incubated

Highest concentration of antibiotic is around the disks, and it decreases as the drug diffuses away

After incubation, the diameters of the zones of inhibition surrounding the disks are measured and compared to standardized values

Read from a standardized chart after zones of inhibition are measured

Broth dilution (MIC)

Serial dilutions of an antibiotic within tubes are performed and then each tube is inoculated with a standard suspension of bacteria and incubated

The lowest concentration of antibiotic that inhibits bacterial growth is the MIC= minimum inhibitory concentration

MICs can be performed in a microtiter plate (and are often automated), but the principle is the same

An aliquot from each tube (or well) without any growth is plated onto an antibiotic-free agar plate and incubated

The lowest concentration of antibiotic that kills 99.9% of bacterial cells (essentially no growth on an agar plate) is the MBC = minimum bactericidal concentration

Performing an MBC can provide information on the “-cidal” vs. “-static” nature of each antibiotic tested

ETest

Mueller Hinton Agar plates are inoculated with a bacterial suspension in a similar manner as for the Kirby-Bauer test

Plastic test strips impregnated with a gradually decreasing concentration of the antibiotic are placed onto the plate and incubated

Multiple strips can be used on a plate; one antibiotic per strip

After incubation, the MIC value is determined where the elliptical zone of inhibition crosses the E-test strip