Title:

The effect of the concentration of the antibiotic Meropenem (1 µg/mL, 4

µg/mL, 16 µg/mL, 64 µg/mL, 256 µg/mL) on the growth of Acinetobacter
baumannii during a 24-period, measured by the minimum inhibitory
concentration (MIC) using the broth macrodilution method

Aim:

SOI: We make decisions for the good of the society based on evidence of small changes that
occur over time.

In the context of this unit’s statement of inquiry, an experiment that is testing how
effectively different concentrations of an antibiotic are able to inhibit the growth of a
bacterium allows for an exploration of the ways in which bacteria have over time developed
antibiotic resistance to better protect themselves. Antibiotic resistance refers to the ability of
bacteria or other microorganisms to develop defense mechanisms against the antibiotics that
were originally effective in killing them (“About Antimicrobial Resistance”). This ability can
develop in a variety of ways such as through mutation, horizontal gene transfer, or the
overuse of the same type of antibiotic (Srakocic). In other words, this experiment is a way to
investigate the idea of evolutionary adaptation. Especially in the world of health and sickness,
small changes are constantly afoot and an antibiotic concentration that was effective a few
years ago, may be harmless to bacteria today. It is only through experiments that carefully
examine the subtle changes that have taken form throughout the years, that it is possible to
make decisions about how current actions taken may need to be modified, and what new
decisions need to be made for the well-being of society.

Background Information:

As the number of bacterial strains and diseases continues to rise, it is necessary to

develop strategies that safeguard peoples’ health and adapt to the evolving landscape.
Acinetobacter baumannii is a type of bacteria belonging to a genus of gram-negative bacteria
called Acinetobacter (Liu). This bacterium is associated with a range of infections, including
pneumonia, bloodstream infections, urinary tract infections, and wound infections
(“Acinetobacter Baumannii Infection”). It is highly contagious and can spread through
contact with contaminated surfaces or simply through person-to-person contact
(Bennington-Castro). The bacterium is also known to be an opportunistic pathogen; it
commonly causes infections in individuals with compromised immune systems whose
resistance against pathogens is already impaired, such as critically ill patients, elderly
individuals, and people with underlying health problems (“Acinetobacter Baumannii
Infection”). As such, Acinetobacter baumannii is a large contributor to hospital-acquired
infections, particularly in intensive care units (Liu).
Along with that, the bacterium has garnered a notorious reputation due to its
resistance mechanisms that have allowed it to become a multidrug resistant bacterium and
due to how rapidly these resistance genes have been spread among the bacteria (Liu). This
antibiotic resistance can be developed in a variety of ways. There can be mutations in the
bacteria that occur from errors in DNA replication either due to substitution, deletion, or
insertion of base pairs that leads to changes in the nucleotide sequence (“Mutations and
selection”). Alternatively, the bacteria can obtain these resistant genes through horizontal
gene transfer where bacteria can transfer their genes to other bacteria of the same kind
through three primary mechanisms: conjugation, transformation, and transduction (“How
Bacteria Build Resistance at the Cellular Level”). These occurrences allow bacteria to fight
against the antibiotic in unique ways such as through biofilm formation, the release of
enzymes, efflux pumps, and more (“How Bacteria Build Resistance at the Cellular Level”).
That said, the chosen antibiotic for this experiment, meropenem, is a broad-spectrum
and intravenous antibiotic belonging to the Beta-Lactam antibiotic class and more
specifically, to a subclass of Beta-Lactam antibiotics called carbapenems (“Meropenem”).
While there are a number of distinctions that can be made between different antibiotic
classes, one of the salient differences is the mechanism of action, i.e., the way in which the
antibiotic kills or inhibits the bacteria (Anderson). Beta-Lactam antibiotics work by
interrupting cell wall synthesis in the bacterial organism. They do this by binding to
penicillin-binding proteins (PBPs) which leads to problems with the cross-linking of
peptidoglycan strands in the cell wall, ultimately resulting in instability in the cell wall, and
lysis of the bacterial cell (Opal).
Carbapenems such as meropenem often cater to more drastic infections when other
antibiotics have failed to inhibit the bacterial growth (Werth). This is because they have been
known to be effective even against multidrug-resistant bacteria and exhibit activity against
aerobic anaerobic, gram-positive, and gram-negative bacteria (Memon). However, it is
equally important to note that while meropenem has shown itself to be a great counter against
many bacteria, the rise of antibiotic resistance has become a troubling topic of discussion
(Sheu). More than ever before, it is crucial to monitor antibiotic resistance patterns to ensure
the continued efficacy of the antibiotics we employ to protect people.
In this experiment, the broth macrodilution method is used to test the susceptibility of
Acinetobacter baumannii to varying concentrations of meropenem. In microbiology and
antimicrobial susceptibility testing, broth dilution is a commonly used and standardized
procedure (Ahmed). In the experiment, the antimicrobial agent is serially diluted in a chosen
growth medium, typically a broth, to create a range of concentrations (Binod). These
concentrations are then inoculated with the bacterium and after incubation, the bacterial
growth in the tubes is compared to determine MIC (Ahmed). Compared to other methods of
antibiotic sensitivity testing, the term “broth macrodilution” underlines the use of liquid
broths as the growth medium and the use of serial dilutions to create the antibiotic
concentrations (Binod).

Research Question:

What is the effect of the concentration of the antibiotic Meropenem (1 µg/mL, 4 µg/mL,
16 µg/mL, 64 µg/mL, 256 µg/mL) (± 5 µg) on the growth of Acinetobacter baumannii
during a 24-hour period measured by the minimum inhibitory concentration (MIC) using
the broth macrodilution method?

Hypothesis:

If the concentration of the antibiotic Meropenem (1 µg/mL, 4 µg/mL, 16 µg/mL, 64

µg/mL, 256 µg/mL) (± 5 µg) increases, then the growth of Acinetobacter baumannii during a
24-hour period using the broth macrodilution method will have a minimum inhibitory
concentration (MIC) of 16 µg/mL. Acinetobacter baumannii and meropenem have been
shown to be extraordinary cases within their groups due to their rare and impressive strength
(Joshi). Acinetobacter baumannii stands out as a highly resilient and versatile Gram-negative
bacterium, regarded for its ability to survive in harsh environments and develop resistance to
several antibiotics (Joshi). On the other hand, meropenem belongs to the carbapenem class of
antibiotics, which are acclaimed for their broad-spectrum activity and effectiveness against
highly resistant bacterial strains (Aslam).
Previous studies, experiments, and papers have shown the MIC for other carbapenem
antibiotics and highly resistant bacteria to fall within similar ranges (Tamma). While this
would typically be considered to be intermediate to high on the MIC spectrum, a slightly
higher MIC is expected given the strength and resistance of Acinetobacter baumannii
(“Microbiology guide to interpreting minimum inhibitory concentration”). Indeed, past
findings suggest that only higher concentrations of carbapenems are able to inhibit the growth
of multidrug resistant bacteria (Nikaido). While there can be inconsistencies and problems
that arise in comparing the MIC number of one antibiotic to another, few studies have fully
explored the efficacy of meropenem on various bacterial strains and other carbapenems share
many similar qualities in their properties and characteristics (“Microbiology guide to
interpreting minimum inhibitory concentration”).
Once the bacterium has obtained the resistance genes whether through heredity,
horizontal gene transfer, a mutation, etc., there are a number of resistance mechanisms that
Acinetobacter baumannii is able to employ to protect itself against attacking antibiotics
(Biggers).
The bacterium can produce enzymes such as beta-lactamases (Munoz-Price).
Beta-lactamases are able to hydrolyze the beta-lactam ring found in many antibiotics which
inactivates the antibiotic and prevents it from binding to the bacterium and exercising its
antimicrobial effects (Munoz-Price). Additionally, they can also produce carbapenemases
which are a subcategory of beta-lactamases that specifically target carbapenems, often
encoded with special resistance genes such as the blaOZA or blaNDM genes (Cui).
Secondly, Acinetobacter baumannii also has efflux pumps, proteins found in the
bacterial cell membrane that protect the bacterium by constantly pumping out harmful
substances such as antibiotics (“How Bacteria Build Resistance at the Cellular Level”). In the
case of Acinetobacter baumannii, it has several efflux pump systems that work in tandem to
transport these unwanted substances out of the cell, including the
resistance-nodulation-division superfamily (Nikaido).
Thirdly, the bacterium can make alterations to the antibiotic targets. Since antibiotics
work by binding to penicillin-binding proteins, part of Acinetobacter baumannii’s resistance
is using new genes to encode altered PBPs that have a lower affinity for binding with
antibiotics (Sharifzadeh). Therefore, this counteracts the effects of the antibiotic and allows
the bacterium to survive even in the antibiotic’s presence (Sharifzadeh). Among others, these
abilities contribute to the high level of antibiotic resistance observed in Acinetobacter
Baumannii and elucidate why if the concentration of the antibiotic Meropenem (1 µg/mL, 4
µg/mL, 16 µg/mL, 64 µg/mL, 256 µg/mL) (± 5 µg) increases, then the growth of
Acinetobacter baumannii during a 24-hour period using the broth macrodilution method will
have a minimum inhibitory concentration (MIC) of 16 µg/mL.

Variables:

Independent and Dependent Variable

Type of Variable Name Method of Manipulation/Measurement

Independent Concentration of To manipulate the independent variable and

Meropenem (µg/mL) achieve the various meropenem concentrations
(1 µg/mL, 4 µg/mL, 16 µg/mL, 64 µg/mL, 256
µg/mL), a four-fold serial dilution is used. A
serial dilution is a stepwise series of dilutions,
where there is a predetermined dilution factor
that remains the same from concentration to
concentration. The method allows you to start
with the greatest concentration and gradually
decrease it by diluting the solution with a
solvent. This facilitates the process of
achieving very small concentrations of the
substance and reduces the uncertainty of the
measurement. The serial dilution is performed
by transferring a small amount of one solution
to a container with more of the solvent, to
dilute the original solution. This dilute sample
is then used as the next base solution to make
an even more diluted solution. In the case of
this experiment, the dilution series starts with a
256 µg solution and the solution decreases by a
factor of 4 with each subsequent tube.
Ultimately, this means that the first tube will be
a 1:4 dilution, the second a 1:16, the third a
1:64, etc.

Dependent Minimum inhibitory The MIC is the dependent variable of the

 concentration (MIC) experiment, however, it will be determined by
assessing the tubes’ turbidity. Turbidity refers
to the cloudiness or haziness of a liquid. It
increases as bacteria multiply and their
population size in the solution grows as they
become more densely packed within the
medium. On the other end of the spectrum, if
the antibiotic effectively inhibits bacterial
growth in a solution, the solution will remain
clear or exhibit a much lower turbidity level.
Turbidity measurement is a convenient and
popular method to monitor bacterial growth as
it is a visual and quantitative indicator of the
presence of bacteria in a solution. It's crucial,
however, to remember that turbidity
measurements don't reveal the precise amount
of bacteria present. Additional approaches,
such as plating and colony counting,
spectrophotometry, or molecular techniques,
may be used for a more precise quantification
or characterization of bacterial growth, but for
such an experiment, it suffices to simply
determine the MIC by visually assessing the
turbidity.

Control Variables

Type of Name Method of Control Possible Effect on Data

Variable

Control Amount of When creating the Maintaining the same amount of broth
Mueller-Hinto meropenem between trials is crucial to ensuring
n broth concentrations, a accurate results. For one, varying
pipette will be used amounts of broth in the solutions can
to measure out of cause problems with the dilution
Mueller-Hinton to series. Since the series relies on
make sure that each diluting the antibiotic in a fixed
tube has exactly volume of broth, having inconsistent
0.75 mL of broth. amounts of broth causes the incorrect
The measurement concentrations to be made as the ratio
 markings on the of antibiotic to broth changes.
pipette will be used Additionally, the amount of broth also
and the liquid will plays a large role in the determination
be fully dispensed of the MIC. This is because more or
from the pipette less broth can make bacteria more or
when transferring to less visible in the solution. For
the tube. Since this example, if a smaller volume of broth
is quite a specific is used in one trial, it may seem like
measurement and there was an excessive amount of
pipettes can be bacterial growth when in reality, it
difficult to measure simply appears that way because with
with, the less broth, the bacteria are
micropipette will be automatically pushed closer together.
used to help control
this variable if
necessary.

Incubator time Each time that the The duration of incubation for each
and incubator is used, all tube directly affects the time available
temperature 30 of the trials will for bacterial reproduction. Not only
be put into the that, but the duration also influences
incubator at the the phase of bacterial growth the tube
same time and taken is in. After incubation, there are four
out at the same time. primary phases that the bacteria will
When incubating go through: lag phase, exponential
the culture dish to phase, stationary phase, and death
grow the phase. If the tubes are incubated for
Acinetobacter varying amounts of time, the
baumannii, this will experiment results are no longer
be for 24 hours and beneficial as any varying amounts of
when incubating the bacteria can be attributed to the
fully prepared tubes, duration of incubation rather than just
this will be for the meropenem concentration. That
18-24 hours. Also, said, the temperature also plays an
the temperature of equally important role as a control
the incubator will variable. The temperature directly
remain at 35°C affects the reproduction rate of the
throughout the bacterium as different bacteria have
entire experiment. diverse optimal growth temperatures
and thus, the temperature in the
incubator can significantly influence
the bacterium’s ability to grow.
 Amount of A micropipette is Changing the amount of bacteria
bacteria in used so that 0.1 mL added to each tube alters the entire
each tube of bacterial composition of the solution. Since
suspension is added some tubes will simply be given a
into each tube. To greater amount of bacteria to start
make sure that there with, their bacterial growth will be
are no tubes with more substantial. However, along with
more or less that, varying amounts of bacteria also
bacteria, completely means varying optical densities. This
empty the can cause significant effects on the
micropipette when data as achieving the 0.5 McFarland
moving from tube to Standard relies on the optical density
tube and carefully compared to the density of the
keep track of which bacterial suspension. This standard is
tubes have already regarded as the norm for antimicrobial
been given the susceptibility testing and culture
suspension. media performance testing which
ensures that the suspension contains
1-2 • 108 colony-forming units (CFU)
per milliliter. Thus, if the amount of
bacteria in each tube isn’t kept the
same, the bacteria won’t be growing
in optimal conditions and the results
won’t be accurate.

Materials:

Item Quantity Size Uncertainty

Test tubes 30x 13 mm x 100 mm -

Sterile cotton plugs 30x - -

Mueller-Hinton 200 mL - -
Broth (MHB)

Incubator 1x - -

Meropenem powder 1g - -

Waste Beaker 1x 100 mL -

Culture of 5 mL - -
 Acinetobacter
baumannii

Sterile cell spreader 1x - -

Inoculation loop 1x - -

Pipette 1x -

Petri dish 1x 8.5 cm diameter -

Disinfectant (70% 50 mL - -
isopropyl alcohol)

Paper towels 20x - -

Marker 1x - -

Gloves 1x - -

Safety glasses 1x - -

Mask 1x - -

Agar 100 g - -

Analytical balance 1x - ± 0.005 mg

Weigh boat 1x - -

Micropipette 1x 10-100 µL ± 0.05 µL

Spectrophotometer 1x - -

Test tube rack 1x 30 slots minimum -

Method:

1. Put on the necessary personal protective equipment for the experiment, namely, safety
glasses, gloves, and a mask.
2. Wipe the table down with disinfectant to make sure that there is a safe environment
for experiment.

Preparing the Bacteria Culture:

3. To inoculate the agar plates, use a sterile inoculation loop to transfer a small amount
of the Acinetobacter baumannii culture onto the agar surface on the petri dish.
 4. Gently spread the culture across the surface of the agar using a sterile cell spreader.
Start from one side of the petri dish and move to the other in a back-and-forth motion,
applying light pressure and covering the entire plate. Continue this streaking process
for at least 30 seconds to ensure that the bacteria are completely and evenly
distributed.
5. Place the inoculated agar plates upside down in an incubator for 24 hours to allow the
bacteria to grow. The incubator temperature should be conducive to the growth of the
bacterium, i.e., the temperature should be around 35°C.
6. After the incubation period, there should be visible growth on the plates.
Acinetobacter baumannii colonies will typically appear as groups of small, round,
pale circles.
7. Using an inoculation loop, gently touch the well-isolated colony of Acinetobacter
baumannii from the fresh culture plate with the loop to transfer a small amount of the
bacteria.
8. Inoculate this bacteria into a tube containing 5 mL of Mueller-Hinton broth.
9. Cover the tube with a stopper and mix the bacterial suspension to achieve a
homogeneous suspension.
10. Adjust the turbidity of the bacterial suspension to obtain an optical density (OD)
equivalent to 0.5 McFarland standard. This ensures that there is a defined number of
bacterial cells in the suspension per mL. This can be done with the help of a
spectrophotometer as is explained below.
a. Calibrate the spectrophotometer and set the wavelength to 600 nm—a
common wavelength for estimating bacterial turbidity.
b. Use a blank cuvette filled with Mueller-Hinton Broth to establish a baseline
for absorbance readings.
c. Take a sample of the bacterial suspension and transfer it to a cuvette.
d. Insert the sample into the spectrophotometer and measure the absorbance.
e. Compare the absorbance reading of the bacterial suspension with the
absorbance values corresponding to the McFarland standards.
f. Adjust the absorbance as necessary by either diluting it with fresh medium or
centrating it with more bacteria, until reaching the desired 0.5 standard.

Preparing Meropenem Solutions:

11. Using a marker, label the 30 tubes with the desired meropenem concentrations: 0
µg/mL, 1 µg/mL, 4 µg/mL, 16 µg/mL, 64 µg/mL, 256 µg/mL. Each concentration
should have 5 tubes labeled so that there are five trials total.
12. Separate the 30 tubes into groups of five tubes with one of each concentration. Carry
steps 13-18 for each group of five tubes.
13. Use the weigh boat and an analytical scale to carefully measure out 256 µg of
meropenem powder. Make sure to tare the weighing container to solely measure the
powder.
14. Use a pipette to measure out 1 mL of Mueller-Hinton Broth in the tubes labeled 256
µg.
 15. Add in the 256 µg of meropenem powder to the tube with the broth and mix until
there is a complete distribution of the antimicrobial agent in the broth.
16. Use a pipette to fill four of the remaining tubes with 0.75 mL of Mueller-Hinton
Broth.
17. Carry out a four-fold serial for the five tubes. This allows you to dilute a highly
concentrated solution to create a less concentrated solution that wouldn't be able to be
accurately obtained otherwise.
a. Transfer 0.25 mg of the 256 µg/mL solution to the tube labeled 64 µg/mL.
b. Thoroughly mix the 64 µg/mL until the solution is homogeneous.
c. Clean the pipette that was used to transfer the solution, or use a new pipette for
the next transfer.
d. Transfer 0.25 mg of the 64 µg/mL solution to the tube labeled 16 µg/mL.
e. Thoroughly mix the 16 µg/mL until the solution is homogeneous.
f. Clean the pipette that was used to transfer the solution, or use a new pipette for
the next transfer.
g. Repeat this process by continuing to transfer 0.25 mg of each solution for each
subsequent tube, using the appropriate dilution factor and volume, until
reaching the 1 µg/mL tube
h. For the 1 µg/mL tube, remove 0.25 mg from the 1 µg/mL into a waste beaker
to maintain the same volume across all the solutions.
18. Measure out 0.75 mL of Mueller-Hinton Broth in the last tube. There should be no
meropenem in this solution as this is the negative control for the experiment that
ensures no other variables are responsible for positive results in the test.

Preparing Bacteria and Meropenem for Incubation:

19. Use a micropipette to add 0.1 mL (100 µg) of the bacterial suspension into all 30 test
tubes.
20. Mix the contents of each tube thoroughly by pipetting up and down several times.
21. Insert a sterile cotton plug into each tube using a gentle twisting motion to inhibit air
movement that may carry microbes.
22. Place all 30 tubes on a test tube rack to hold them upright while they are in the
incubator. Several racks can be used if necessary.
23. Clean up. Materials such as test tubes and pipettes. Other materials such as the agar
and paper towels can be disposed of in the waste bin. Materials that have been
contaminated with the bacterium such as the culture dishes need to be diluted in
bleach and then thrown away in a sealed biohazardous bag.
24. Disinfect the table with the disinfectant solution and wash hands.

Incubation and Interpretation:

25. Incubate all 30 tubes at 35-37°C for 18-24 hours.
26. After incubation, visually inspect each tube for the presence or absence of bacterial
growth by looking at the turbidity (cloudiness) of the tubes.
 27. Determine the MIC of the bacterium, noting that the cloudier the solution is, the
greater the bacterial growth was. The minimum inhibitory concentration (MIC) is
defined as the lowest concentration of meropenem that inhibits all visible growth of
Acinetobacter baumannii. This means that starting from the tube with the lowest
concentration, the MIC is the first tube that is completely clear.

Photo of Experimental Setup:

Figure 1. Example of set up for broth macrodilution experiment with six trials before incubation

Safety Considerations:

While there is a level of risk when handling pathogenic or potentially dangerous strains of
bacteria, this risk can be considerably reduced with the right safety precautions and
procedures in mind. Since the safety risks in the experiment largely stem from dangers with
contamination from bacteria, standard aseptic techniques should be followed (Stephens).
Personal protective equipment should be worn during the experiment, most importantly
safety goggles, gloves, and a mask, to reduce the likelihood of direct contact or exposure to
the bacterium. Parts of the experiment that necessitate handling of the bacteria should be
carried out in a biosafety cabinet or some other containment device to best prevent the release
of bacteria to the surrounding environment (Varma). Finally, the work surface should be
regularly disinfected, equipment should be sterilized (flame sterilized if possible), and hands
should be regularly washed (“Aseptic Techniques”). Following these safety precautions
should minimize the risk of exposure and create a safe environment for conducting the
experiment.

Ethical/Environmental Considerations:
The ethical and environmental consideration for the experiment primarily relates to the
handling and disposal of the bacterium. Have a plan in place to take action in the event of an
accidental spill or leak by using proper disinfectants and absorbent materials to effectively
clean up the spill. Also, follow proper storage of the bacterium during and after the
experiment. Any container with bacteria such as culture dishes should be wrapped with
sealing film and stored upside down to inhibit contamination (Scoville). Finally, the bacteria
should be thoughtfully disposed of; before throwing it away, soak the used culture tubes and
petri dishes in a diluted solution of bleach (“Guideline for working with bacterial cultures”).
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