chapter 13, Antimicrobials and Resistance

alexander flemings findings (1928)

there were zones of inhibitions around mold, and staph aureus did not grow. the mold juice was antibiotics

where do antibiotics come from (2)

in the wild: antibiotics produced by some bacteria and fungi

• Though something has antibiotic properties it doesn't mean we call that material an antibiotic (tree bark)

in the lab:  man- made derivatives of natural compounds

• Synthetic cmps need to be approved to be used. 

how do antibiotics work?

Antibiotics inhibit bacterial growth/ reproduction or survival

• Can prevent growth without killing the cell

• Either inhibit growth (accessory) or inhibit survival (required)

ex//

• DNA gyrase is accessory

• Cell wall synthesis is necessary

bacteriostatic

• describing action of antibiotics.

• prevent bacterial growth/ reproduction, but DOESNT KILL BACTERIA

• ex// blocking protein synthesis, inhibiting DNA replication

• Added antimicrobial, bacteria doesnt change

• Removed antimicrobial, bacteria continues growing

bactericidal

• describing action of antibiotics

• antibiotics KILLS BACTERIA

• ex// destroying cell walls, disrupting membranes

• Added antimicrobial, kills bacteria

• Removed antimicrobial, bacteria is still dead

antibiotic resistance

 if a microbe gains ability to resist effects of an antibiotic

types of (antibiotic) resistance (2)

1. intrinsic resistance

2. acquired resistance

intrinsic resistance (peniciliin ex// w gram -ve +ve)

• Some bacteria are naturally resistant to antibiotics

• Don't need to evolve to resist the activity of an antibiotic

ex// gram -ve bacteria are more resistant to penicilin than gram +ve bacteria

• Penicillin targets peptidoglycan

• gram +ve bacteria PG are more exposed than gram -ve

• gram -ve bacteria have an outer membrane barrier & thin PG layer

• Decrease uptake, increased efflux

• Broad range, not specific to one type of antibiotic (targets outer layer of cell)

• Works against have activity across a range of species

• Gram -ve and +ve

Antimicrobials that attack intracellularly are more specific than the ones that attack peptidoglycan. PG is more conserved across all species whereas intracellularly there can be different things inside

acquired resistance

• Some bacteria become resistant through mutation/evolution and natural selection for features that are less susceptible to antibiotics

• Happens over generations

ex// Antibiotic erythromycin targets the ribosome → inhibits protein synthesis

• Bacterial ribosome evolves over 1000s of years so that erythromycin can no longer bind to the ribosome

• Bacteria become resistant to antibiotic

• Mutation for something more targeted (narrow spectrum of activity)

• Target alterations, inactivating enzymes from inside the cell

• Gram -ve or +ve

Antimicrobials that attack intracellularly are more specific than the ones that attack peptidoglycan. PG is more conserved across all species whereas intracellularly there can be different things inside

how do bacteria become drug resistant?

• population has some bacteria that are drug resistant

• Antibiotics added, the non-resistant bacteria is knocked out

• The niche at the infection is colonized by the drug resistant bacteria and is taken over. Selecting microbes. Speeding up natural selection

where does resistance come from

mutation —> evolution

• Change target of the antibiotic in bacteria

• Alter metabolic pathways

• Change in membrane permeability

ex//

• change enzyme in cell to catalyze better

• modifying protein for DNA binding protein (transcription)

• changing membrane permeability: (stop expressing transporter proteins and start producing efflux pumps that pump antimicrobials outside of the cell)

• express inactivating enzymes that are embedded in the structures in the cell wall

ways of testing antibiotic sensitivity (2)

1. disk diffusion assay

2. minimum inhibitory concentration (MIC)

disc diffustion assay

Plate-based assay to test for antibiotic susceptibility

Steps:

1. Take isolated pathogen from patient

2. Grow pathogen in pure culture after 48h

3. Inoculate agar plate with bacteria

4. Spread bacteria around evenly

5. Place disc impregnated with known concentration of antibiotic you are testing onto agar (each number is a different antibiotic)

6. Antibiotics diffuse into agar, preventing growth of susceptible bacteria. This is incubated for 24-48h

7. Measure zone of inhibition: the area around the antibiotic disc in which bacteria cannot grow. Observe how well antibiotic inhibited growth. measure in cm or mm

   1. Bigger zone of inhibition, better antibiotic, more susceptible to antibiotic

   2. No zone, microbe is completely resistant to that antibiotic

Closer bacteria can get to disk, more resistant the bacteria is to that antibiotic. If a bacterium cannot get close to the disk, then the antibiotic kills/hurts that bacterium that means the bacterium is susceptible to that antibiotic

Resistant: smaller than threshold (smaller disk)
Susceptible: higher than threshold (bigger disk)

minimum inhibitory concentration (MIC)

Liquid medium-based assay to test for antibiotic susceptibility. In lab

Steps:

1. Dispense antibiotics, in gradient, into 96 well plate

• Each row = different antibiotic

• Each column = different Concentration

One side is only bacterial growth medium

One side is only growth control, 0 antimicrobial

• All rows should have bacterial growth

Lowest conc that inhibited bacterial growth is the minimal inhibitory concentration MIC 

Liquid assay to test for antibiotic

susceptibility

Steps:

2. Inoculate each well with same volume of bacterial culture

3. Incubate plate to allow bacteria to grow

4. MIC is lowest concentration of antibiotic where you see NO bacterial growth

lower MIC= more susceptible, weaker resistance

higher MIC= more resistant (stronger resistance)