Antibiotic Resistance of Escherichia coli
 
 

Since the 1940s, researchers have discovered numerous naturally occurring and synthetic antibiotics.  And, as we have mentioned in lecture, bacteria that are resistant to all of these antibiotics have been observed.  In many cases, a given bacterium may exhibit multiple drug resistance; that is, it is resistant to several different antibiotics.  Often, these antibiotic resistance genes are located on plasmids and can be transferred from one cell to another and even from one bacterial species to another.  As a result, it is not unusual for normal bacteria isolated from the body to exhibit resistance to one or more commonly used antibiotics.

In order to further investigate the genetic diversity of our Escherichia coli isolates, and to determine the prevalence of antibiotic resistance of E. coli among members of the Davidson College community, we will examine the resistance of our individual E. coli isolates to several common antibiotics.

Day 1:  Grow an overnight liquid culture of your bacteria.  Using sterile technique, transfer a colony of E. coli from your EMB agar plate to a 15mL conical tube containing approximately 5-10ml of tryptic soy broth.  Incubate overnight at 37^oC with shaking.

Day 2:  Plate bacteria on a 100 mm Petri plate containing Mueller Hinton agar.  This medium routinely is used in clinical settings for performing antibiotic susceptibility tests.  Add 200 microliters of your overnight culture to a Petri plate.  Using a cell spreader, spread the bacteria across the entire surface of the plate.  Note: for this experiment, we want to produce a confluent lawn of bacteria; we are not streaking for isolated colonies.

After spreading the bacteria across the plate, add a series of antibiotic discs to the plate.  We have discs of filter paper that have been soaked in common antibiotics at the following concentrations:
 

Using forceps, place one disc of each antibiotic at a different location on the plate.  Gently tap down the discs with forceps.  The discs are marked to differentiate them, but it might be helpful to mark on the plate which disc was placed in which location.

Incubate the plate overnight at 37^oC.

Day 3:  Remove your plate from the incubator and observe the bacterial growth.  Measure and record the zone of inhibition (in mm) surrounding each disc.  According to the National Committee for Clinical Laboratory Standards, the following chart should be used to interpret your data (measurements are in mm):
 

As you complete this experiment, consider the following questions:

1.  How important is the concentration of antibiotic that is used?
2.  Why are we using 3 times as much tetracycline as, for instance, streptomycin?
3.  For an unknown antibiotic, how would determine the optimal concentration?
4.  How could you isolate mutants of your E. coli strain that exhibit altered resistance?
5.  How could you determine if the antibiotic resistance exhibited by your E. coli is caused by chromosome-encoded or plasmid-encoded genes?
6.  Why, when you have a bacterial disease (such as strep throat), do physicians remind you to take all of the provided antibiotics, even if the symptoms disappear?