Antibiotics

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 http://www.pbs.org/wgbh/frontline/film/hunting-the-nightmare-bacteria/

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

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

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.

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).

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

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

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

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

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

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

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.

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

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

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.

Disk Diffusion Assay Cont.

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.

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

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.

Antibiotic Resistance Classifications Susceptible – zone of inhibition ≥ 26 mm Intermediate – zone of inhibition 23-25 mm Resistant – zone of inhibition ≤ 22 mm Clinicians use these types of classifications to choose appropriate antibiotics to combat a particular infection

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  

Lab Day 3 Good example Bad Example

Antibiotic Resistance

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

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

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

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

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?

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

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.

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?

Use your lab data to evaluate Model 2

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

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

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.

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.

The Rest of the Story

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

Lateral Gene Transfer

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

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?

The evolution of bacteria on a mega agar plate! 1000 1 10 100 100 10 1 https://www.youtube.com/watch?v=plVk4NVIUh8

Wrap up TED Talk: What do we do when antibiotics don’t work anymore? https://www.ted.com/talks/maryn_mckenna_what_do_we_do_when_antibiotics_don_t_work_any_more#t-1006929