Ulaş Erdem BARUT / Antibiotic susceptibility profile of DSM9751 + and – QxyA / 19.08.

2022

ABSTRACT

Epsilometer test (e-test) is one of the tests performed to measure antibiotic resistance in
bacteria and to measure Minimum Inhibitory Concentration (MIC). In this experiment, an e-
test was applied to reveal the antibiotic resistance profiles of Pseudomonas saponiphila DSM
9751 strain with plasmid and Pseudomonas saponiphila 9751Δ strain without plasmid. Strains
grown in liquid and reaching an OD 600 of about 0.3-0.5 were then grown in Mueller Hinton
Agar plate. After the cells were seeded, e-testing was performed with 18 different antibiotics
with different concentrations for each strain and MIC (μg/mL) values were determined. While
it is expected that the strain with the plasmid will be more resistant, it has been observed that
this situation only occurs when Doxycycline is used. In some antibiotics used, clear zone was
formed and cells could not grow, while in some, clear zone was not formed. The reason why
the zone does not occur is either because they are resistant to antibiotics or because the range
of antibiotic concentration used is insufficient.

INTRODUCTION

Antibiotics, which have been used quite frequently by people recently and given by doctors,
are antimicrobial agents used against bacteria. They are used to fight bacterial infections and
can inhibit the growth and spread of bacteria or even kill them. While there are quite a few
[1]
different types of antibiotics, they all have different functions and places of action .
Antibiotic sensitivity tests can help find out which antibiotic will be most effective in treating
the infections. These tests also help find out which substances can be used against
microorganisms that are resistant to antibiotics. Antimicrobial resistance arises when
microbes such as fungi or bacteria develop the ability to defeat drugs designed to kill them.
[2]
Thanks to this event, bacteria or related microbes cannot be killed and continue to grow .
When microorganisms with antimicrobial resistance survive and reproduce, they have
resistance properties in their DNA that can spread to other bacteria [3].

The Kirby-Bauer Disk Diffusion Susceptibility Test, which is one of the methods used to
monitor antimicrobial resistance, is very easy to perform. An antimicrobial agent of known
concentration is added to the filter paper placed on the agar. After a while, this substance
[4]
begins to diffuse into the surrounding agar . After a certain time, two conspicuous areas
appear on the plate. The first area is known as the clear zone and bacterial growth cannot be
observed in this area due to the growth-inhibiting and lethal effects of the antibiotic. Bacteria
continued to grow in areas outside the clear zone because the antibiotic used was outside the

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Ulaş Erdem BARUT / Antibiotic susceptibility profile of DSM9751 + and – QxyA / 19.08.2022

[5]
area of influence . Figure 1 shows a plate was created using different antibiotics and the
resulting areas.

No growth area

Growth area

Figure 1: Disk diffusion test for S. aureus [6].

Epsilometer test (E-test), which is one of the new methods for antimicrobial resistance
control, has more detailed usage stages. The instrument used consists of a predefined
antibiotic concentration gradient on a rectangular plastic. When an inoculated agar is placed
on the plate, the antibiotic in the strip begins to diffuse onto the agar in a gradient fashion. As
in the disk diffusion test, while clear zones are the areas created by bacteria that cannot grow
thanks to antibiotics, other parts are areas that are not affected by the concentration of the
antibiotic used. The MIC can be read directly from the scale at the point where the edge of the
inhibition ellipse intersects the MIC test strip [7, 8]. The MIC referred to is defined as the lowest
antimicrobial concentration that will inhibit the visible growth of a microorganism after
incubation. Figure 2 shows an example of an e-test and the meaning of the markings on the
strip used for the test.

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Ulaş Erdem BARUT / Antibiotic susceptibility profile of DSM9751 + and – QxyA / 19.08.2022

Figure 2: E-test for MIC determination. Image of the test and schematic representation of the e-test for
interpretation from left to right, respectively [9].

The mediums to be used for susceptibility tests also have a special importance. Mueller
Hinton agar is generally used when performing antibiotic tests. While beef extract and casein
it contains provide essential nutrients such vitamins nitrogen, sulfur, starch is added to absorb
any toxic metabolites produced [10].

The aim of this experiment is to reveal the resistance profiles and MIC (µg/ml) values of
Pseudomonas saponiphila DSM 9751 and Pseudomonas saponiphila DSM9751Δ strains by e-
test with 18 different antibiotics with different concentrations.

MATERIALS & METHODS

Materials

In this experiment, Pseudomonas saponiphila DSM 9751 and DSM 9751Δ, graduated
cylinders, 50 ml falcon tubes, petri dishes, Bunsen burner, cotton buts, pH meter,
spectrophotometer (Shimadzu UV-160), bottles, flasks, 250 ml erlenmeyers, eppendorfs,
autoclave, shaker and e-test with strips with different concentrations of 18 different antibiotics
(Amoxicillin, Azithromycin, Benzylpenicilin, Chloramphenicol, Ciprofloxacin,
Clarithromycin, Clindamycin, Doxycycline, Enrofloxacin, Erythromycin, Kanamycin,
Levofloxacin, Norfloxacin, Ofloxacin, Sulfamethoxazole, Tetracycline, Trimethoprim, and
Vancomycin) were used. In addition, Mueller Hinton Broth, 1.5% Agar and M63B1 were
used as medium.

Methods

First, 500 ml of water is put into two 1-liter bottles. Then, 10.5 grams of Mueller Hinton, 7.5
grams of agar (1.5%) were added to prepare Mueller Hinton agar. For the growth of cells in
liquid culture, 10 ml of Brunner medium was placed in 2 falcons. How much BAC to add to
these falcons were found from the formula 10000 * x/10=200 and 200 ml of BAC was added
to both falcons. Then, 100 microliters of DSM9751 samples were added to these falcons. 1ml
of MH broth was put into ependorf. After a row of samples was taken from the plate with
DSM9751Δ with cotton but, these samples were homogeneously distributed in 1 ml
eppendorf and vortexed. All vortexed samples were added to 50ml MH in 250ml flask. A
flask containing only MH was placed next to this sample for control purposes and kept in the
shaker at 110 rpm. The absorbance value was controlled until 0.5. Control process was done

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Ulaş Erdem BARUT / Antibiotic susceptibility profile of DSM9751 + and – QxyA / 19.08.2022

in Shimadzu UV-160 branded spectrophotometer at 600 nm wavelength. MH agar plates were

prepared for e-testing on both DSM9751 and DSM9751Δ samples. After autoclaving, 2
bottles of 500 ml each, which were prepared before, were poured on the plates as 25 ml, and a
total of 40 plates were prepared and waited for approximately 20 minutes for them to dry. In
order to perform e-test on the prepared plates, the samples were diluted 1/10 and placed. For
this, 100 microliters from DSM9751 and DSM9751Δ samples and 900 microliters from MH
Broth were transferred into ependorf. Later, a sample of this eppendorf was taken with cotton
buts, filtered and streaked onto the plates. After all the streak processes were completed, e-test
strips containing different antibiotics at different concentrations were placed slowly with fired
tweezers and the cells were allowed to grow and the results were photographed under a
magnifying glass.

This experiment was repeated in 63B1 media. The medium was prepared by adding 160
microliters of BAC into the 63B1 media prepared as in the protocol. Afterwards, the 500ml
media, which was ready, was filled in 250ml flasks by adding 50ml each. 50 microliters of
DSM9751 sample taken at 4°C were taken, vortexed and placed in liquid media and allowed
to grow for 2 days. After 2 days of growth, 1 ml of DSM9751+BAC+63B1 sample was taken
and the OD value was measured at 600nm. Then, for the preparation of DSM9751Δ sample, a
row was taken from the plate with cotton butt and spread in ependorf containing 1ml of MH
broth. Afterwards, all samples in ependorf were added to the flask in 50ml MH Broth and
allowed to grow. 100 microliters from 9751 sample grown for 2 days and 900 microliters
from MH Broth were taken and 1/10 dilution was made and placed in 4 ependorfs.
Afterwards, samples were taken from cotton butt and ependorf, streaked onto MH agar plate,
and 18 different e-tests were performed. On the other hand, since the DSM9751Δ samples that
were left to grow grew too much, 100 microliters were taken from the very dense flask and
placed in 50ml MH broth to make a 1/500 dilution and allowed to grow again. After
measuring OD at 600nm every 3 hours, the grown samples were diluted 1/10 and streaked on
the plate like DSM9751 and e-test strips were placed one by one.

RESULTS & DISCUSSION

The aim of this experiment is to observe the antibiotic susceptibility of P. saponiphila DSM
9751 and 9751Δ strains and to observe what differences occur in the strain without plasmid.
As a result of the study, while no clear zone is formed in some antibiotics, it has been
observed that some clear zones are formed and are not resistant to a certain extent. Stored

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Ulaş Erdem BARUT / Antibiotic susceptibility profile of DSM9751 + and – QxyA / 19.08.2022

strains were cultured until they reached an absorbance value of approximately 0.3. Then, e-
test was performed with 18 different antibiotics at different concentrations. MIC (µg/ml)
values and list of different samples of antibiotics are shown in Table 1.

Table 1: MIC (µg/ml) values of DSM 9751 and DSM 9751Δ strains given as 1-MIC and 2-MIC. In addition, the
first column shows which antibiotics the MIC values belong to and the classification of antibiotics. DSM9751
represents the sample with plasmid, while the DSM9751Δ represents the sample without the plasmid.

The reason why 2 different MIC values are shown in Table 1 is that the DSM 9751Δ strain
used in the first part of the experiment was cultured too much and the OD 600 value obtained in
the spectrophotometer was much higher than 1. According to the researches, it has been seen
that it reduces the number of excess cell number effective number of antibiotics, and therefore
the experiment was repeated [11].

When the plates and Table 1 are examined, it does not show any clear zone formation in
Macrolides, Beta-lactams, Lincosamides, Glycopeptides type antibiotics and trimethoprim.
From this, it can be said that the concentration values shown on the strip are not sufficient for
the interpretation of antibiotic resistance. When benzylpenicillin is examined, there is no clear
zone in all samples. However, this interpretation can only be made up to 256µg/ml
concentration of the antibiotic. It is not known whether resistance occurs at a 500µg/ml
concentration of the antibiotic because the value on the strip remains less.

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Ulaş Erdem BARUT / Antibiotic susceptibility profile of DSM9751 + and – QxyA / 19.08.2022

On the other hand, samples with plasmids are expected to have greater resistance than
samples without plasmids, because the antibiotic resistance property usually comes with the
[12]
plasmid . However, according to Table 1, the interpretation that the strain with plasmid is
more resistant can only be made when doxycycline is used. While the MIC value was
96μg/mL in the strain with plasmid, it was seen that the strain without plasmid was 12μg/mL
and the strain with plasmid was more resistant.

In other antibiotics, MIC values of the sample without plasmid in general were higher. Since
there is a direct ratio between MIC and antibiotic resistance, it can be interpreted that their
[13]
resistance is higher . When Table 1 is examined, it is seen that some MIC values do not
change depending on whether there is a plasmid or not. For example, when the antibiotic
tetracycline and its effects were examined, it was seen that the MIC value was 12μg/mL for
both samples. As specified by Table 1 , it can be interpreted that having a plasmid is not
related to providing resistance to tetracycline.

As a result, it is not known whether P. saponiphila DSM 9751 and DSM 9751Δ strains have
resistance to the antibiotics used. In some types of antibiotics, clear zones are formed and
cells cannot grow, while in others, no clear zones are formed. The no clear zone may be either
because they are resistant to antibiotics or it is because of the insufficient range of
concentrations on the e-test strip used. When strains with and without plasmids are compared,
it is seen that the strain with plasmid is expected to have more antibiotic resistance, but this
only happens when Doxycycline is used.

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