Microbe Case Study attached other, Jimmy, got 1 Part I – Measuring Resistance Katelyn was excited to start her summer job in her microbiology professor’

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Microbe Case Study attached other, Jimmy, got
1

Part I – Measuring Resistance
Katelyn was excited to start her summer job in her microbiology
professor’s research laboratory. She had enjoyed Dr. Johnson’s
class, and when she saw the fyer recruiting undergraduate lab
assistants for the summer, she had jumped at the opportunity.
She was looking forward to making new discoveries in the lab.

On her frst day, she was supposed to meet with Dr. Johnson
to talk about what she would be doing. She knew the lab
focused on antibiotic resistance in Staphylococcus aureus, espe-
cially MRSA (methicillin-resistant S. aureus ).

by
Maureen Leonard
Biology Department
Mount Mary College, Milwaukee, WI

Antibiotic Resistance:
Can We Ever Win?

NATIONAL CENTER FOR CASE STUDY TEACHING IN SCIENCE

stants!Johnson to grab a spot today!

(You must have taken Biology 200 Microbiology to apply)

Summer semesterDr. James Johnson

antibiotic resistanc

hi li h h f ll f h j l

We’re looking for undergraduate lab assiIf yes, e-mail Dr.

Sciences Building 1Johnson@ ictionaluniv.edu

Interested in studying microbiale?

Do you want to work in a research lab? Are you interested in bacteria?

Have you heard of antibiotic resistance?

She still remembered the scare her family had last year when her little br ttle brother, Jimmy, got
so sick. He’d been playing in the neighborhood playground and cut his lip when he fell of the jungle
gym. Of course he always had cuts and scrapes—he was a fve-year-old boy! Tis time though his lip swelled up and he
developed a fever. When her mother took him to the doctor, the pediatrician said the cut was infected and had prescribed
cephalothin, an antibiotic related to penicillin, and recommended fushing the cut regularly to help clear up the infection.

Two days later, Jimmy was in the hospital with a fever of 103°F, coughing up blood and having trouble breathing. T e
emergency room doctors told the family that Jimmy had developed pneumonia. Tey started him on IV antibiotics,
including ceftriaxone and nafcillin, both also relatives of penicillin.

It was lucky for Jimmy that one of the doctors decided to check for MRSA, because that’s what it was! MRSA is
resistant to most of the penicillin derivatives. Most cases of MRSA are hospital-acquired from patients who are already
susceptible to infection, but the ER doctor explained that community-acquired MRSA was becoming more common.
Te doctor then switched the treatment to vancomycin, a completely diferent kind of antibiotic, and Jimmy got better
quickly after that.

Katelyn had dropped Jimmy of at swimming lessons just before coming to work at the lab. As she waited in the
hallway for Dr. Johnson, she hoped that she would be at least a small part of helping other people like Jimmy deal
with these scary resistant microbes. She was surprised when the professor burst out of the lab, almost running into her.

“Hi Katelyn, I’m really sorry but I have to run to a meeting right now—they sprung it on me last minute. Tere are a
bunch of plates in the incubator right now that need their zones of inhibition measured. I’ll be back in a few hours,”
Dr. Johnson said as he rushed down the hallway with a stack of folders.

Katelyn dug out her old lab notebook to look up what she was supposed to do. She found the lab where she and her
fellow students had examined the antimicrobial properties of antibiotics using the Kirby-Bauer disk dif usion tech-
nique. Looking at the plates Dr. Johnson had told her about, she saw they had all been “lawned,” or completely coated
with microbes to make a thick hazy layer over the agar surface. She could also see paper disks with letters on them,
and some of the disks had clear zones around them where the microbe had been inhibited (Fig. 1). Her notebook
explained how to measure the zone of inhibition around the disks (Fig. 2).

“Antibiotic Resistance” by Maureen Leonard Page 1

Plate 1.

Plate 2.

Plate 3.

S. aureus

PE

CEME

VA

S. aureus

PE

CE
ME

VA

S. aureus

PE

CE
ME

VA

PE

CEME

MRSA

VA

PE

CEME

MRSA

VA

PE

CEME

MRSA

VA

Figure 1. Agar plates of S. aureus or MRSA lawns with antibiotic disks placed on them.

“Antibiotic Resistance” by Maureen Leonard Page 2

NATIONAL CENTER FOR CASE STUDY TEACHING IN SCIENCE

Inhibition
(clear) zone

Measure in mm

Figure 2. Katelyn’s diagram of how to measure a zone of inhibition from her microbiology lab notebook.

Exercise1

Measure the zones of inhibition for each antibiotic on the plates shown in Figure 1 and note the measurements in the
spaces in Table 1 below. (Note: Te Kirby-Bauer method is standardized so that no zone of inhibition is scored as a 0,
and all others include the disk as part of the zone.)

Key: PE = penicillin, ME = methicillin, CE = cephalothin, and VA = vancomycin

Plate S. aureus MRSA

1

PE

ME

CE

VA

2

PE

ME

CE

VA

3

PE

ME

CE

VA

An average, or mean (x), is a measure of central tendency in the data, or what value occurs in the middle of the data
set. Te mean is calculated by adding up all the values for a given set of data, then dividing by the sample size (n).

n

x i
Average x i 1

n
Standard deviation measures the spread of the data—as in how variable the data set is. Te standard deviation (s) is
calculated by the following:

“Antibiotic Resistance” by Maureen Leonard Page 3

NATIONAL CENTER FOR CASE STUDY TEACHING IN SCIENCE

( x x)
2

Standard deviation s
n 1

Standard error measures the diference between the sample you have taken and the whole population of values. T e
standard error (SE) is calculated as follows:

s
Standard error SE

n

Exercise 2

In Table 2 below calculate and record the averages and standard errors for each antibiotic in S. aureus and MRSA.

S. aureus MRSA

Average SE Average SE

PE

ME

CE

VA

Exercise 3

Now, redraw Tables 1 and 2 into a single, more organized table. Be sure to label the table appropriately.

“Antibiotic Resistance” by Maureen Leonard Page 4

NATIONAL CENTER FOR CASE STUDY TEACHING IN SCIENCE

Exercise 4

Graph the results from Table 2. Be sure to label the fgure and the axes correctly.

“Antibiotic Resistance” by Maureen Leonard Page 5

NATIONAL CENTER FOR CASE STUDY TEACHING IN SCIENCE

Questions

1. What do you think the experimental question is?
2. What hypotheses can you come up with to answer the experimental question?
3. If your hypothesis is correct, what would the plates look like (i.e., what predictions would you make for each

hypothesis)?
4. Is the experiment you just collected data for an appropriate test of the experimental question you came up with

in your answer to Question 1?
5. Which antibiotics where most ef ective against S. aureus? Against MRSA?
6. When comparing the antibiotics efective against both, were there diferences in ef ectiveness?
7. What other questions do the data shown in Figure 1 make you think of?

“Antibiotic Resistance” by Maureen Leonard Page 6

NATIONAL CENTER FOR CASE STUDY TEACHING IN SCIENCE

Part II – Resistance
Among the frst antibiotics used on a large scale was penicillin, which was discovered in 1929 by Alexander Fleming. It
was fnally isolated and synthesized in large quantities in 1943. Penicillin works by interfering with the bacterial cell
wall synthesis. Without a cell wall, the bacterial cells cannot maintain their shape in changing osmotic conditions. T is
puts signifcant selective pressure on the microbes to evolve, as they cannot survive the osmotic stress. Any microbe
that can resist these drugs will survive and reproduce more, making the population of microbes antibiotic resistant.

T e specifc mechanism of penicillin is the prevention of cell wall synthesis by
the -lactam ring of the antibiotic (Fig. 3), which binds and inhibits an enzyme
required by the bacterium in this process.

Te enzyme is called penicillin-binding protein (PBP), even though it is an enzyme
involved in cell wall synthesis. Normally enzymes have names that indicate what

Figure 3. T e -lactam ringthey do and end in the sufx -ase, like lactase, the enzyme that breaks down lactose.
common to the penicillin

Figure 4 is a representation of PBP and its active site. family of antibiotics.

Active site

Figure 4. PBP (penicillin-binding protein) active site is a groove allowing formation of
cross-links in the bacterial cell wall.

Bacterial cell walls are layered structures, where each layer is made of peptidoglycan, a sugar and protein polymer. Each
layer is cross-linked to the next, strengthening the wall and allowing the cell to resist osmotic pressure. Te way the
enzyme PBP works is to form those cross-bridges by joining strings of amino acids together in the active site, which is
a groove in the protein (Fig. 5).

Cross-bridge
PBP

Peptidoglycan Amino acids
layers

Figure 5. Cross-link formation in bacterial cell walls by PBP (penicillin-binding protein).

“Antibiotic Resistance” by Maureen Leonard Page 7

NATIONAL CENTER FOR CASE STUDY TEACHING IN SCIENCE

Te PBP takes amino acid residues attached to peptidoglycan layers and forms
bridges between them within the active site groove. Tis cross-linking, or
cross-bridging, stabilizes and strengthens the cell wall. -lactam antibiotics
interfere with the PBP enzyme by binding to the active site, blocking the site
from the amino acids (Fig. 6).

Figure 6. Inhibition of PBP

NH

O

Tere are over 80 natural and semi-synthetic forms of -lactam antibiotics,
(penicillin-binding protein) by

including cephalothin and methicillin. Vancomycin also interferes with cell
-lactam blocking the active site.

wall synthesis, but its mechanism of action is to bind directly to the cell wall
components (Figs. 7 and 8).

PBP

a. Normal PBP binding and cross-bridge formation

+ =

b. PBP inhibited by -lactam antibiotic

c. Cell wall does not form properly

Figure 7. PBP (penicillin-binding protein), the enzyme that allows the bacterial cell
wall to form cross-bridges, is inhibited by the -lactam family of antibiotics. T is
prevents proper cell wall synthesis and the bacterium will succumb to osmotic stress.

“Antibiotic Resistance” by Maureen Leonard Page 8

NATIONAL CENTER FOR CASE STUDY TEACHING IN SCIENCE

PBP

a. Normal PBP binding and cross-bridge formation

+

Vancomycin

b. Vancomycin binds PBP substrate

c. Cell wall does not form properly

Figure 8. Vancomycin inhibits cell wall synthesis a diferent way by binding PBP’s
substrates and preventing cross-bridging. Tis prevents proper cell wall synthesis
and the bacterium will succumb to osmotic stress.

T e frst MRSA case was discovered in 1961 in a British hospital, and was the result of a mutation in the enzyme
normally inhibited by the -lactam ring of methicillin. Te site where the antibiotic would bind no longer allowed
access to the ring, so the enzyme continued to function normally. Te microbe acquired a new gene that, when made
into protein, was a diferent version of PBP, one that couldn’t be inhibited by penicillin.

Questions

1. Describe what is happening in Figures 7 and 8 in a complete sentence of your own words.
2. What are the diferences in how -lactam antibiotics and vancomycin work?
3. What other mechanisms might arise to allow resistance to the -lactam antibiotics?
4. Could resistance arise to vancomycin? Why or why not?

“Antibiotic Resistance” by Maureen Leonard Page 9

NATIONAL CENTER FOR CASE STUDY TEACHING IN SCIENCE

Part III – Restoring Susceptibility
Katelyn had been working for Dr. Johnson for a month, and while she had become quite good at measuring inhibition
zones, she didn’t know why she was doing all this work. She had gotten very curious after she began doing all the
measurements on a new set of antibiotics. Tis experiment involved infecting mice with MRSA and tracking how the
MRSA grew over time.

Data were collected by counting the cells of MRSA taken from fuid samples from the mice. Te cells were measured
by taking one gram of the fuid and spreading it over plates, but now Katelyn counted the colonies that grew on the
plate after 24 hours. Because there were so many, she actually measured the colonies as “log CFU/g.” A CFU is a
colony forming unit, or essentially a cell that will divide into a colony that can be seen. Because there can be so many,
Katelyn measured them on a logarithmic (log) scale. Te raw data in her lab notebook looked like the following:

Table 1. Efect of treatment on MRSA in mice after 24 hours of drug treatment as log CFU/g.
Treatment

FtsZ inhibitor +
Trial Control FtsZ inhibitor Imipenem imipenem

1 9.11 7.55 6.98 2.21
2 8.25 8.12 8.12 4.55
3 9.05 9.27 9.01 7.98
4 9.37 8.02 8.33 5.64
5 8.80 7.65 7.64 1.25
6 9.25 8.3 7.77 9.98
7 9.41 7.99 8.21 6.78
8 9.11 7.71 7.98 3.45
9 8.61 8.22 7.68 2.45

10 9.12 8.11 8.21 1.01

Questions

1. What do you think the experimental question is?
2. What hypotheses can you come up with to answer the experimental question?
3. What predictions would you make for each hypothesis?
4. Looking at the data in Table 1, what do these numbers mean? (Keep in mind a log value means each integer

increase is actually a ten-fold increase in the number of cells.)
5. What do you think FtsZ inhibitor and imipenem are?

Next, Katelyn further analyzed the data she collected by calculating the average and standard error.

Table 2. Average efect of treatment on MRSA in mice after 24 hours of drug treatment (log CFU/g).

Treatment

FtsZ inhibitor +
Control FtsZ inhibitor Imipenem imipenem

Average 9.008 8.094 7.993 4.53
SE 0.114 0.153 0.169 0.954

Question

6. Does Table 2 change your interpretation of the experimental data from Question 4? Why or why not?

“Antibiotic Resistance” by Maureen Leonard Page 10

NATIONAL CENTER FOR CASE STUDY TEACHING IN SCIENCE

She then made the following graph (Fig. 9):

Figure 9. Efects of treatments on MRSA numbers in mice. Samples were taken at 24 hours
post-infection. (Figure modifed from Tan et al. 2012).

Katelyn was very excited by the results, but she didn’t know what an FtsZ inhibitor was, or what imipenem was. She
decided to ask Dr. Johnson what his research was all about.

“Dr. Johnson, look at these results I got from the last round of plates,” Katelyn said as she handed him a copy of the
results above. “What exactly are we testing here?”

Dr. Johnson looked at the results and smiled. “Tese are great! Tis could really change the way we deal with antibiotic
resistance.

“To answer your question, -lactam antibiotics are still the most heavily used antibiotics, though resistance is a big
problem. Most treatments have changed to using multidrug regimens in the hopes of allowing the antibiotic to still
function while at least slowing down the resistance mechanism.

“Another approach involves looking for other proteins that could be inhibited, and looking for existing inhibitors to
make into drugs. Instead of looking just for new antibiotics, we’re looking for new targets.”

Dr. Johnson handed Katelyn a few papers to read. In them she learned that the protein, FtsZ, helps “pinch of ” the
new cells at the end of cell division. Tis involves interacting with the cell wall as it is synthesized, and if FtsZ is
interfered with, cell wall synthesis stops too. Tis prevents cell division and the microbe can no longer reproduce.

Dr. Johnson tested the new target idea by using a recently discovered inhibitor of FtsZ to see what efects that had on a
MRSA infection. As part of the study, the inhibitor was tested by itself and in combination with imipenem, a -lactam
antibiotic, resulting in the data above.

Questions

7. How efective was the FtsZ inhibitor alone? Imipenem alone?
8. How efective was the combination of the inhibitor and the -lactam antibiotic?
9. How would you explain these results?

10. What questions would you pursue next?


Case copyright held by the National Center for Case Study Teaching in Science, University at Buffalo, State University of New York. Origi-
nally published November 30, 2012. Please see our usage guidelines, which outline our policy concerning permissible reproduction of this
work.

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