1. Antibiotics

2. Superbugs
One example of a “superbug” is Methicillin-resistant Staphylococcus aureus (MRSA). MRSA is a bacteria that is resistant to many antibiotics.
Frontline Video: Hunting the Nightmare Bacteria

3. Class Discussion Why is the issue of antibiotic resistance important?
Personal stories?
Student Worksheet on MRSA

4. Discovery of Antibiotics
In 1928, Alexander Fleming discovered the first antibiotic.
A fungus (Penicillium)
inhibited the growth of…
A bacterium (Staphylococcus)
Schematic illustrating Fleming’s discovery of antibiotics

5. What are Antibiotics?
Some organisms make and secrete chemicals that inhibit the growth of other organisms.
These chemicals are called, “antibiotics,” meaning “against life.”
We use antibiotics to stop bacterial infections in humans.

6. How do Antibiotics Work?
To be useful to humans, the antibiotic must have selective toxicity.
It must prevent the growth of some organisms (bacteria) but not harm or prevent the growth in other organisms (humans).

7. Criteria for Antibiotic Targets
Target needs to be a structure, enzyme or chemical pathway that humans do not have.

8. Return to Worksheet Cell membrane Cytoplasm Cell Wall Pili Ribosomes
Put Labels on
Flagellum
Chromosome
Plasmid

9. What bacterial cell structures might be good targets for antibiotics?
Check Points
What bacterial cell structures might be good targets for antibiotics?

10. Possible Targets Examples: Cell wall synthesis
Protein synthesis – bacterial ribosomes are different from human ribosomes
DNA replication – enzymes are different.

11. How does our antibiotic nalidixic acid work?
Inhibits DNA replication
DNA polymerase,
polymerizes DNA
DNAP
DNAP
DNA gyrase causes and repairs breaks in the DNA to relieve the strain on the DNA strands as they are unwound.
DNA gyrase,
Assists in unwinding double stranded DNA

12. How does our antibiotic, nalidixic acid, work?
Inhibits DNA replication
nalidixic acid,
binds DNA gyrase
DNA polymerase,
polymerizes DNA
DNAP
DNAP
DNA gyrase,
Assists in unwinding double stranded DNA

13. How does our antibiotic, nalidixic acid, work?
nalidixic acid binds to DNA gyrase.
The double stranded DNA cannot be unwound. DNA replication stalls.
The chromosome fragments.
End result: Bacteria dies.
Here is the full story: DNA gyrase makes and repairs nicks in the DNA to relive the tension as the DNA strands are unwound. When nalidixic acid binds to DNA gyrase, a stable enzyme DNA complex is formed which initially prevents the DNA from being unwound and leads to the replication fork stalling. Breaks in the DNA are then made but not repairs causing the chromosome fragmentation.

14. Review What are antibiotics? Where do they come from?
How does our antibiotic, nalidixic acid, work?

15. Day 1 Lab Demo Good example Bad Example
Note: A single micro centrifuge tube is used throughout this procedure.
Good example Bad Example

16. How can we test if an antibiotic is effective at killing bacteria?
Use a Disk Diffusion Assay
A paper disk is infused with an antibiotic.
The antibiotic spreads out (diffuses) through the agar all around the disk.
The concentration of antibiotic is strongest closest to the disk, and gets less and less as you get further from the disk.
In the image this is illustrated with blue dye instead of antibiotics.

17. Disk Diffusion Assay Cont.

18. Antibiotic Resistance Terms
Susceptible:
The bacteria are inhibited by the recommended dose of the antibiotic.
Bacterial resistance is absent.
The antibiotic is an appropriate choice for treating the infection.

19. Antibiotic Resistance Terms
Intermediate:
The bacteria are partially inhibited by the antibiotic.
These antibiotics can be used in body sites where the drug is concentrated (ex. urinary tract).
These antibiotics may be used if a higher concentration of the antibiotic is safe

20. Antibiotic Resistance Terms
Resistant:
The bacteria is NOT inhibited by the recommended dose of the antibiotic.
Indicates the bacterial likely have resistance mechanisms to the antibiotic
The bacteria is not killed by the antibiotic, even at higher doses.
The antibiotic is not an appropriate choice for treating that bacterial infection.

21. Antibiotic Resistance Classifications
Susceptible – zone of inhibition ≥ 26 mm
Intermediate – zone of inhibition mm
Resistant – zone of inhibition ≤ 22 mm
Clinicians use these types of classifications to choose appropriate antibiotics to combat a particular infection

22. Return to Lab Handout Make predictions:
Draw where the bacteria will grow if a population of susceptible bacteria is swabbed onto the plate and the disk has antibiotics
Draw where the bacteria will grow if a disk with no antibiotic is placed on the plate
Draw where the bacteria will grow if a population of intermediate bacteria is swabbed onto the plate and the disk has antibiotics
Draw where the bacteria will grow if a population of resistant bacteria is swabbed onto the plate and the disk has antibiotics

23. Lab Day 3
Good example Bad Example

24. Antibiotic
Resistance

25. Learning Targets
What strategies do bacteria use to fight nalidixic acid?
Where do these strategies come from?

26. Antibiotic Resistance
Different bacteria use different resistance strategies.
Some bacteria use multiple strategies.
Number of mutations accumulated

27. E. coli Resistance Strategies
Strategy 1: Actively pump antibiotic out of cell.
DNA Level: Mutation that causes over-expression of gene for pumps  more efflux pumps in cell membrane.
nalidixic acid
Cell Level:
Efflux pumps

28. E. coli Resistance Strategies
Strategy 2: DNA gyrase shape changes slightly and antibiotic can no longer bind to it.
DNA Level: Mutation in region of DNA gyrase which binds nalidixic acid
nalidixic acid
Cell Level:
modified
DNA gyrase

29. Summary Check Point
What are the two strategies E. coli uses to fight against antibiotics?
Where did these strategies come from?
How is there variation in bacterial resistance levels?

30. Record your data, and the class data on the student handout
Keep the lid on your plate. On the bottom side of the plate, draw a line through the center of the zone with a marker and mark the edges of the zone

31. Record your data Cont.
NOTE: especially for the day 3 plates, the edge of the zone may have a faint inner ring of growth. If this occurs, use the inner ring as the edge of the zone of no growth.

32. Use your lab data to evaluate Model 1
What do the different colored circles represent?
What is happening to the starting population on day 1 in the top row? In the bottom row?
What is the difference between the top and bottom rows in this model?
Does the model fit with the class data you collected from the lab? Why or why not?

33. Use your lab data to evaluate Model 2

34. Claim Evidence Reasoning (CER)
Use the CER method to answer the question:
How and why do antibiotics become useless?
Make a claim (A statement that answers the above question)
Support your claim with evidence (use anything from this unit, background information, lab data, the models)
Explain all parts of the evidence, why is this evidence significant, then make a judgement that is supported by your evidence and evaluation of the evidence

35. Useful Words
As you think about the models, these words may be helpful:
Population
Individual
Trait
Variation of a trait
Initial Variation
Advantageous variation of a trait (adaptation)
Environmental change
Random Mutation

36. Natural Selection
Natural selection means that some organisms survive and have more offspring because they have a trait that is advantageous in that particular environment.

37. Words that may be helpful as you think about making a general model of natural selection:
A population is a collection of organisms in a species living in a specific area.
Example: All the moths living in forests in New England are a population of moths.
Example: All the Red-Bellied Black Snakes living in Australia are a population of Red-Bellied Black Snakes.
An individual is one organism in a population.
An individual moth is one moth.
An individual Red-Bellied Black Snake is one snake.
Traits. Here are some examples:
Wing color in moths.
Weight of moths.
Ear size of rabbits.
Hair color of humans.

38. The Rest of the Story

39. Plasmids Plasmids have genes for antibiotic resistance.
Pilus promotes cell-to-cell contact
Plasmid carries nalidixic acid resistance gene (qnr)
Tra machinery can transfer plasmid to other bacteria
Chromosome

40. Lateral Gene Transfer

41. E. coli Resistance Strategies
Strategy 3: QNR protein binds DNA gyrase and prevents antibiotic binding
DNA Level: acquire plasmid with qnr gene
nalidixic acid
Cell Level:
QNR
DNA gyrase

42. Summary Plasmids reproduce and move into surrounding bacteria.
Process is called lateral gene transfer.
How does lateral gene transfer make antibiotic resistance an even BIGGER problem?

43. The evolution of bacteria on a mega agar plate!
1000 1 10
100
100 10 1

44. Wrap up TED Talk: What do we do when antibiotics don’t work anymore?