M465:Antibiotic Resistance

Antibiotic Resistance

Antibiotics are molecules that kill bacteria or inhibit their growth. Common antibiotics target important cellular functions in bacteria (such as cell wall remodeling or protein synthesis). Believe it or not, many microbes are naturally resistant to antibiotics, either because they produce them or because that resistance is advantageous in the habitat they occupy. One place in which such selection is currently taking place, is in the human microbiome. Unfortunately, due to the overuse of antibiotics (both in medicine and agriculture), widespread antibiotic resistance has become the norm. The figure below captures the trend and shows the increasing prevalence of antibiotic resistance in human pathogens.

Today you will be testing whether or not your honey bee isolates are resistant to antibiotics. Honey bees are agricultural organisms and are treated with the antibiotic tetracycline prophylactically, twice a year, by many bee keepers. Because of this practice, the honey bee microbiome has tetracycline resistance determinants. For a nice paper on these determinants, check this link out [1]

You will be using a soft agar overlay technique to test the degree of resistance in your bacterial isolates. First, you will take a cellulose disk, impregnated with antibiotics, and put it in the center of your agar plate. You will then take 2 mL of molten agar (45C water bath) and add 100 uL of an overnight culture of your isolate to that 2mL *very quickly* by inversion two times. Pour your soft agar over the surface of your agar plate, nutating the plate in order to spread the soft agar evenly. Don't take too long or the agar will solidify and you will not have time to pour it!

PROTOCOL

Set up an overnight culture the day before! For each isolate, perform the steps below.

• 1. Grab a 1.5 mL tube filled with cellulose disks impregnated with antibiotic. You will need three, one for each antibiotic we are testing.
  2. Label an agar plate of the appropriate medium with your isolate and the antibiotic you are testing.
  3. Using sterile tweezers, grab a single disk from the 1.5 mL tube and place it at the center of your agar plate.
  4. Grab a tube of melted agar from the 45C bath.
  5. Add 100 ul of your overnight culture of your isolate to the melted agar.
  6. Vortex briefly to mix the bacteria into the soft agar and immediately pour onto the appropriate agar plate.
  7. Tilt your agar plate back and forth to distribute the agar evenly over the surface.
  8. After the agar hardens (about 10 minutes), invert the plate and incubate overnight at 37C under appropriate atmosphere.
  9. Results should be visible in the next few days. Come in to check on your experiment and if you see growth in the agar lawn, take your plate out of the incubator and measure the zone of inhibition around the antibiotic disk.