SYNOPSIS
In this three-week lab, students examine the evolution of antibiotic resistance in a common gut
bacterium, Escherichia coli. They isolate E. coli from their own bodies and learn the techniques
for growing these bacteria in the lab and then screen them for resistance to commonly used
antibiotics. They also look at how long it takes a population of E. coli to lose antibiotic resistance
in a lab environment where there is no advantage to being resistant to any antibiotic.
OBJECTIVES
INTRODUCTION
This lab will use antibiotic resistance as an example on what evolutionary biologists do and how
they do it.
PART 1: Introduction to study system and developing hypotheses and research methods
PART 2: Data collection Testing for resistance and loss of resistance
PART 3: Data collection and analyses and presentation preparation
When posted, download the following papers from the CTools course site and read them.
1) Levy, S.B. March 1998. The challenge of antibiotic resistance. Scientific American. Pp. 46-53.
2) Nesse, R.M. and Williams G.C. November 1998. Evolution and the Origins of Disease.
Scientific American. Pp. 86-93.
Other articles are optionalthey report both studies to measure the extent of antibiotic
resistance and factors that may contribute to it.
Bonten, M., Stobberingh, J. P., Houben, A. 1992. Antibiotic resistance of Escherichia coli in
fecal samples of healthy people in two different areas of an industrialized country. Infection 20:
258-262
Erb, A., Sturmer, T. Brenner, H. 2007. Prevalence of antibiotic resistance in Escherichia coli:
overview of geographical, temporal, and methodological variations. Eur. J. Clin. Microbiol. Dis.
26:83-90.
Manning, S. D., Pearlman, M. D., Tallman, P., Pierson, C. L., Foxman, B. 2001. Frequency of
Antibiotic Resistance among Group B Streptococcus Isolated from Healthy College Students.
Clinical Infectious Diseases 33:137-139.
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Aiello, A.E., E.L. Larson, S.B. Levy. 2007. Consumer antibacterial soaps: Effective or just risky?
Clinical Infectious Diseases 45(Supplement): S137-S147.
Khachatourians, G.G. 1998. Agricultural use of antibiotics and the evolution and transfer of
antibiotic resistant bacteria. Canadian Medical Association Journal 159: 1129-1136.
Lloyd, D.H. 2007. Reservoirs of antimicrobial resistance in pet animals. Clinical Infectious
Diseases 45(Supplement): S148-S152.
Background: Evolutionary biologists ask "why" rather than "how". Causation: ultimate (why?)
versus proximate (how?). A why question is one that asks the historical sequence of
causation. A how question asks about the present day functional sequence.
Which of the above responses best addresses the historical events that led to the existence
of antibiotic resistance as a trait? (which is a why question and which is a how
question?).
What are antibiotics and where do they come from?
How do antibiotics work?
How can bacteria be resistant to (= insensitive =not affected by) antibiotics?
Origins of antibiotic resistance genes in bacteria?
Where do resistance genes originally come from?
What are all the possible ways in which an individual bacterium can pick up a resistance
gene?
Why is Levy concerned with the increasing use of antibacterial soaps?
What are the ways in which agricultural uses of antibiotics may impact human health and
the evolution of antibiotic resistance in general?
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PROTOCOL
Background: Escherichia coli is a common bacterium in the gut of many organisms. For this
lab, we will use sterile cotton swabs to isolate E. coli from ourselves. The swabs will be used to
inoculate Petri dishes containing a selective and differential medium, eosin-methylene blue
(EMB). Using this medium, we can select for and differentiate E. coli from other bacteria based
on two properties. First, EMB supports the growth of gram-negative organisms and inhibits the
growth or gram-positive organisms. Second, E. coli can be differentiated from other coliform
bacteria by its metallic green sheen when grown on EMB (many other bacteria will have a pink
mucousy appearance). Once inoculated, the plates are incubated at 37C (body temperature)
to support growth of the bacterial colony.
Part 1 Objectives: Working individually, isolate individual colonies of E. coli for use next week
in antibiotic resistance assay.
Fill out the survey on personal history answers will remain anonymous by use of the same
code number you used on your plate. Later, we will correlate personal history with antibiotic
resistance in recovered bacteria.
Carry out a practice dilution- dilute the E. coli sample as directed. You will compare turbidity
of the culture (absorbance) with actual plate counts. This is practice for part 2.
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Part 2 Objectives:
Test strains cultured from last week for antibiotic resistance.
Discuss possible follow-up independent experiments. These will be restricted to the
types of information that can be gleaned from survey data collected in first week of this
3-week lab.
o Determine the approximate titer of the cell culture (109 cells/ml if fully turbid) and the
dilution required to get to countable numbers of colonies on the plates.
o Set up the dilution using sterile saline and microcentrifuge tubes
o Plate 100 l from the target dilution plus one dilution above and below the target
o Plate onto each type of antibiotic plate plus a control plate (no antibiotic).
o Label with group name and dilution factor
o Give the plates to your GSI for incubation.
o You will examine the plates next week to compare colony numbers on the plates with
antibiotic vs. those without.
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