1.2.3 Attack of the Superbug

Attack of the Superbugs

MI 1.2.3

Goals

• Describe the three ways that genes can be transferred between bacteria

• Model gene transfer between bacterial cells to create an antibiotic-resistant bacteria

• Demonstrate proper aseptic technique

History

• Before the discovery of antibiotics, there was no effective treatment for infectious diseases (e.g. pneumonia, gonorrhea, strep throat, skin infections, etc.).
• What was the first antibiotic? Who discovered it?

• Hospitals were filled with people experiencing sepsis from a minor cut or scratch - all we could do was wait and hope for the best.

History

• By 1941, penicillin was being mass produced and it changed the course of medicine.

• Prior to the development of antibiotics, the #1 cause of death was infections. Now, infectious disease doesn't even make it to the top 3.

History

• Antibiotics were given freely, even when patients were known not to have bacterial infections. Why?

• Although antibiotic resistance has always existed, it has skyrocketed sine the 1990s.

When a bacterium is resistant to antibiotic, how does it prevent the antibiotic from working?

There are two categories of antibiotic resistance:

Intrinsic Resistance vs. Acquired Resistance

When a bacterium develops resistance to an antibiotic that it was previously sensitive to. This always occurs due to genetic changes. These genetic changes can be due to: (1) spontaneous mutations (2) gene transfer/recombination between bacteria. Gene transfer (recombination) can occur in 3 ways: 1. Transformation 2. Conjugation 3. Transduction This type of resistance is MUCH more serious than intrinsinc resistance.

AKA "natural" resistance. This is the resistance that a bacterium naturally has due to its typical structure. E.g. gram negative bacteria are naturally more resistant to antibiotics th an gram positive bacteria because of their lipid outer membrane. The outer membrane in gram negative cells makes it harder for antibiotics to penetrate and enter the cell.

How is resistance acquired? - Gene Transfer!

Bacterial Recombination Lab

You will work with 2 strains of E. coli. E. coli I contains a gene found on chromosomal DNA that encodes for streptomycin resistance. E. coli II contains a gene found on a plasmid that encodes for ampicillin resistance. Your goals in this multi-day lab will be to: 1. Confirm resistance of each strain to their respective antibiotic. 2. Prepare a "mating" plate where recombination (gene transfer) can happen.3. Test for recombination.

Day 1

Inoculate 4 agar plates with both strains of E. coli. 1. plan LB agar 2. LB agar + streptomycin 3. LB agar + ampicillin 4. LB agar + streptomycin + ampcillin. Discuss: Based off of what you know about each strain's resistance, which plate(s) would you expect to see growth of each strain?

Strain I - streptomycin resistance Strain II - ampicillin resistance

Day 2

View growth from day 1 plates to confirm correct antibiotic resistance.Create a "mating" plate

• Inoculate 1 plain LB agar plate with both E. coli strains (& mix them together) to provide an environment for recombination to occur

Day 3

Inoculate 3 different agar plates with the bacterial growth from day 2's "mating" plate.1. LB agar + streptomycin 2. LB agar + ampicillin 3. LB agar + streptomycin + ampicillin Discuss: If recombination was successful, what plate(s) should show growth? Which of these plates confirms recombination?

Inactivation with Enzymes

• Some bacteria produce enzymes that are able to cleave (chop up) certain types of antibiotics.

• For example, many bacteria produce an enzyme known as beta lactamase. This enzyme is able to chop up all beta lactam antibiotics (penicillins and cephalosporins).

The first antibiotic was penicillin and it was discovered by Alexander Fleming.

Expelling the antibiotic

Some bacteria have special protein channels in their cell membranes, called efflux pumps, that pump the antibiotic out of the cell immediately after it enters.

Cell wall and Membrane Impermeability

• Some antibiotics are simply unable to get into the bacterial cell due to their cell wall structure or proteins within their membrane that prevents the antibiotic from entering.
• E.g. Gram negative bacteria

Changing the target

• All antibiotics target a specific structure within the bacterial cell in order to either kill it or prevent it from reproducing.
• If the structure is changed in some way, that will affect the antibiotic's ability to bind to the target which will render the antibiotic ineffective.