The Resistance Profile of Bacteria Salmonella Sp Towards
Antibacterial Ethanol Extract of Bidara Leaves
(Ziziphus Mauritiana)
Fitriyati Mukhlishoh
1
, Pratikah Verdianti
1
, Dwi Krihariyani
2
1
Departement of Forensics Science, Postgraduate School, Universitas Airlangga, Jl. Airlangga 4-6, Surabaya, Indonesia
2
Study Program of Medical Laboratory Technology, Poltekkes Kemenkes Surabaya, Jl. Karangmenjangan 18A, Surabaya,
Indonesia
Keywords: Typhus, Salmonella sp,bacteria resistance, ethanol extract of leaves bidara (Ziziphus mauritiana), dilution.
Abstract: Typhus is an infection of the digestive tract which remains one of the health problems in Indonesia.
Sanitation is a cause of the spread of the infection of Salmonella sp. Parts of plants such as leaves, stems,
and roots are usually used as sources of treatment, especially for treating the typhus without prescription.
Some Indonesian people consume the water of bidara leaves (Ziziphus mauritiana) to treat the disease. It
can be explained by previous studies that the bidara leaves contains tannins and alkaloids which can prevent
the growth of bacteria. The purpose of this experiment is to determine the resistance of Salmonella sp
towards antibacterial ethanol extract of bidara leaves. The bacteria Salmonella sp was isolated from the
blood sample of typhus patients on the extract in the Laboratory of Bacteriology. Antibacterial ethanol
extract of bidara leaves had been tested with liquid dilution method consisted of six concentrations, namely
30%, 25%, 20%, 15%, 10% and 5%. The result of the MBC value from five Salmonella sp obtained from
five different patients showed a significantly different. The extract of bidara leaves on sample 1, 2, 5 have
the MBC value 15%; whereas in samples 3 and 4 have higher bactericides, namely at a concentration of
10%.
1 INTRODUCTION
Typhoid fever or known as typhus is the infection on
the digestive tract. Infection on the alimentary canal
caused by Salmonella sp specifically Salmonella
typhi is remain existed as a common health problem
among Indonesian people (Paputungan, 2016).
Moreover, in the case of typhus, Indonesia is in
the third rank and WHO reported that the number of
the sufferers increase each year in the approximate
800 per 100.000 people. This infection-related
disease is spread widely in all Indonesian areas with
the similar number of cases in each area
(Yuswananda, 2015).
Previous studies showed that children aged
between 6-12 (Umah, 2014) or between 3-19
(Nelwan, 2012) are at the highest risk for the
potential of the typhus. Currently, this condition is
worsened by the resistance of the bacteria
Salmonella sp. Earlier in 1981, Sippel, et. al.,
reported that Salmonella sp resists to
chloramphenicol (Hammad, et. al., 2011). The
resistance appeared due to the use of unproperly
anticiotic, for example, the take of a heavy dose of
antibiotic (Febiana, 2012).
Bacterial resistant on the patients can worsen
their poor health condition because it indicates that
they need more doses of antibiotic drugs and the
longer period is have to be taken in consuming the
antibiotic. Meanwhile, the long-term of antibiotic
consumption will finally defect the digestive system
(Utami and Puspaningtyas, 2013).
The report of Salmonella sp resistance towards
the chemical antibiotic became the baseline for us to
find new antibacterial; another viable alternative
from plants as an antibiotic resource without
negative side effects on the human body especially
on the digestive canal. Plants as natural resources
can be taken as either prevention or treatment
because plants contain many active compounds
which function as antibacterial, antioxidant, anti-
inflammation and immunomodulator (Karon, et. al.,
2011). Further, work by Karon, et. al., (2011) proves
Mukhlishoh, F., Verdianti, P. and Krihariyani, D.
The Resistance Profile Of Bacteria Salmonella Sp Towards Antibacterial Ethanol Extract Of Bidara Leaves (Ziziphus Mauritiana).
DOI: 10.5220/0007543403810385
In Proceedings of the 2nd International Conference Postgraduate School (ICPS 2018), pages 381-385
ISBN: 978-989-758-348-3
Copyright
c
 2018 by SCITEPRESS – Science and Technology Publications, Lda. All rights reserved
381

that ethanol extract of Bidara leaves (Ziziphus
mauritiana) demonstrates a positive response in the
prevention of the growth of some bacterias:
Salmonella typhi, Salmonella paratyphi,
Staphylococcus aureus, and Vibrio cholera.
In addition, the Bidara leaves are utilized
empirically by group of people in Lenteng village,
Sumenep Regency, to treat common fever and
typhus. However, the consumption of the leaves is
regardless of the scientific knowledge about the
active compounds contain inside the leaves; as a
consequence, they traditionally sublime the leaves
without knowing the proper composition. Ironically,
Bidara as wild plants were cropped so that recently it
is difficult to find Bidara trees around people’s
house even for some people who are taking the
leaves to treat the family member who is suffering
from common fever or typhus.
Because of the resistance of bacteria of
Salmonella sp in antibiotics has been reported and
also people haven't known the benefits of plants
such as bidara leaves, thus this research is a
continuity of the research that has been done by
Karon et al. (2011). The research by Karon et al.,
was about the antibacterial resistance of ethanol
extract of bidara leaves (Ziziphus mauritiana)
against Salmonella typhi bacteria and Salmonella
paratyphi ATCC. Therefore, this research will use
Salmonella sp bacterias which isolated from the
blood of typhoid fever patients because ATCC
bacteria may not be able to describe the bacteria that
attack the community so that the resistance profile of
the Salmonella sp bacteria in the blood of typhoid
fever patients in that extract will be known.
2 METHOD
Post-test through experimental design was used to
examine the antibacterial power of Bidara leaves’
ethanol extract towards Salmonella sp bacteria by
using liquid dilution method. We took Salmonella sp
bacteria which was isolated from the blood sample
of typhus patients. It is done because the typhus
caused by ATCC bacteria had not been concluded
yet that it is which infected the patients so that the
resistance profile of Salmonella sp bacteria on the
typhus patients’ blood to the antibacterial compound
of the extract would be known.
2.1 Extraction Substance
The Bidara leaves were gotten from Lenteng Village
which is located in Sumenep Regency, Madura.
Firstly, the leaves were dried and sublimated until it
obtained 250 gram of the leaf powder. Secondly, the
powder was extracted by using ethanol solvent 95 %
through the maceration method; the maceration
process was running within 5 days by changing the
solvent every day. Thirdly, filtrate was collected
every 24 hours of immersion in which each filtrate
was mixed and evaporated by rotary evaporator at
40° C and 120 rpm of speed so that we gained 100
% of ethanol extract of Bidara leaves.
The used of concentration were 30 %, 25 %, 20
%, 15 %, 10 %, and 5 %. Following were the
method of obtaining the extract through each
concentration:
2.1.1 Concentration 30 % : 30 μl bidara
leaves extract 100 % + 70 μl DMSO
2.2.1 Concentration 25 % : 25 μl bidara
leaves extract 100 % + 75 μl DMSO
2.3.1 Concentration 20 % : 20 μl bidara
leaves extract 100 % + 80 μl DMSO
2.4.1 Concentration 15 % : 15 μl ml bidara
leaves extract 100 % + 85 μl DMSO
2.5.1 Concentration 10 % : 10 μl bidara
leaves extract 100 % + 90 μl DMSO
2.6.1 Concentration 5% : 5 μl bidara leaves
extract 100 % + 95 μl DMSO.
Ciprofloxacin antibiotic was used for the positive
control with concentration 30 %, 25 %, 20 %, 15 %,
10 %, and 5 %. These were the method to modulate
the expected concentration of ciprofloxacin:
2.1.1 Concentration 30 % : 0,03 gram tablet
of ciprofloxacin + 100 μl sterile aquades
2.2.1 Concentration 25 % : 0,025 gram tablet
of ciprofloxacin + 100 μl sterile aquades
2.3.1 Concentration 20 % : 0,02 gram tablet
of ciprofloxacin + 100 μl sterile aquades
2.4.1 Concentration 15 % : 0,015 gram tablet
of ciprofloxacin + 100 μl sterile aquades
2.5.1 Concentration 10 % : 0,01 gram tablet
of ciprofloxacin + 100 μl sterile aquades
2.6.1 Concentration 5 % : 0,005 gram tablet
of ciprofloxacin + 100 μl sterile aquades
2.2 Bacterial Suspension Preparation
In this research, there were two kinds of bacterial
suspensions that been used. One was Salmonella sp
which was isolated from the blood sample of typhus
patients placed as the experimental group. This
suspension consisted of 5 suspensions in which the
bacterias were collected from 5 different patients.
And the other one was bacteria suspension of
Salmonella sp ATCC which was placed as a positive
and negative control. The bacterial suspension was
ICPS 2018 - 2nd International Conference Postgraduate School
382

made by mixing the bacteria colony that grew on the
media Nutrient Agar Slant (NAS) into sterile 2 ml
PZ, then the turbidity was equated to the standard
turbidity of McFarland 0,5.
2.3 Liquid Dilutin Method
Each extract solvent with concentration 30 %, 25 %,
20 %, 15 %, 10 %, and 5 % was inoculated with 5
bacteria Salmonella sp suspensions from the blood
sample of patients, where each blood sample was
added one loop of bacteria suspension. The similar
procedure was also implemented to the positive
control with the Salmonella sp ATCC bacteria
suspension for each ciprofloxacin solvent. In
different from that, for the negative control was used
sterile aquades to be inoculated with one loop of
Salmonella sp ATCC bacteria suspension.
After completing the inoculation with bacterial
suspension, then each was incubated into an
incubator at 37 °C during 24 hours. After 24 hours,
it was observed to know the presence or the absence
of the turbidity in each tested solvent. Continually,
the subculture of tested-mixed solvent was
undertaken to conclude whether the bacteria grew or
not by using bacteria suspension which had
incubated on the media Mueller Hinton Agar (MHA)
by a strict method. The incubation was arranged at
37 °C for 24 hours.
The Minimum Bactericidal Concentration
(MBC) was determined from the lowest
concentration which can reduce the viability of the
bacteria; whereas the Minimum Inhibitory
Concentration (MIC) was determined by the lowest
level of the bacterial agent that inhibits the growth
(Vineethe, et. al, 2015).
2.4 Data Analysis
The data were analyzed by Kruskal Wallis statistical
method to know the difference of resistance profile
on Salmonella sp bacteria in the blood of typhus
patients towards the antibacterial of Ziziphus
mauritiana leaves’ ethanol extract.
3 FINDINGS AND DISCUSSION
These are the laboratory test results of antibacterial
ethanol extract of Bidara leaves towards Salmonella
sp bacteria compared to positive control and
negative control.
Tabel 1: Laboratory test result of antibacterial extract of Ziziphus mauritiana leaves on Salmonella sp in the patients’ blood
sample using liquid dilution method
C
R
1
2
3
4
5
Ct
(+)
Ct
(-)
T
S
T
S
T
S
T
S
T
S
T
S
T
S
30%
R
1
-
-
-
-
-
-
-
-
-
-
-
-
+
+
R
2
-
-
-
-
-
-
-
-
-
-
-
-
R
3
-
-
-
-
-
-
-
-
-
-
-
-
R
4
-
-
-
-
-
-
-
-
-
-
-
-
25%
R
1
-
-
-
-
-
-
-
-
-
-
-
-
R
2
-
-
-
-
-
-
-
-
-
-
-
-
R
3
-
-
-
-
-
-
-
-
-
-
-
-
R
4
-
-
-
-
-
-
-
-
-
-
-
-
20%
R
1
-
-
-
-
-
-
-
-
-
-
-
-
R
2
-
-
-
-
-
-
-
-
-
-
-
-
R
3
-
-
-
-
-
-
-
-
-
-
-
-
R
4
-
-
-
-
-
-
-
-
-
-
-
-
15%
R
1
-
-
-
-
-
-
-
-
-
-
-
-
R
2
-
-
-
-
-
-
-
-
-
-
-
-
R
3
-
-
-
-
-
-
-
-
-
-
-
-
R
4
-
-
-
-
-
-
-
-
-
-
-
-
10%
R
1
+
+
+
+
-
-
-
-
+
+
-
-
R
2
+
+
+
+
-
-
-
-
+
+
-
-
R
3
+
+
+
+
-
-
-
-
+
+
-
-
R
4
+
+
+
+
-
-
-
-
+
+
-
-
5%
R
1
+
+
+
+
+
+
+
+
+
+
-
-
R
2
+
+
+
+
+
+
+
+
+
+
-
-
The Resistance Profile Of Bacteria Salmonella Sp Towards Antibacterial Ethanol Extract Of Bidara Leaves (Ziziphus Mauritiana)
383

R
3
+
+
+
+
+
+
+
+
+
+
-
-
R
4
+
+
+
+
+
+
+
+
+
+
-
-
MBC
15%
15%
10%
10%
15%
5%
MIC
10%
10%
5%
5%
10%
Table Information:
C = Concentration
T = Turbidity
+ : Turbid
- : Unturbid
S = Sublimation on the media MHA
+ : bacteria are grown
- : bacteria are not grown
The test group 1, 2, 3, 4, 5 were using
Salmonella sp bacteria which isolated from the
patients’ blood sample and bidara leaves’ ethanol
extract by using each concentration
Ct (+) = Positive control contains ciprofloxacin with
concentrations which were inoculated by the
bacteria Salmonella sp ATCC
Ct (-) = Negative control contains sterile aquades
that was inoculated by the bacteria Salmonella sp
ATCC
MBC = the Minimum Bactericidal Concentration
MIC = the Minimum Inhibitory Concentration
3.1 Statistical Test Result
Table 2: The result of Kruskal Wallis statistical method
The data on the table are interpreted by these
guidelines:
If the significance value (P) ≥ α, then H
0
is accepted
and H
1
is rejected
If the significance value (P) < α, then H
0
is rejected
and H
1
is accepted
The table shows that p-value is 0.046 and α is
0.05. In comparison p < α which means H
0
is
rejected and H
1
is accepted. Hence, there is a
difference on the resistance profile of bacteria
Salmonella sp to Bidara leaves’ ethanol extract.
3.2 Discussion
There are different findings resulted from blood
sample of typhus patients where some
concentrations used are 30 %, 25 %, 20 %, 15 %, 10
% and 5 %. On sample 1, 2, and 5 the bacteria could
be grown at concentration 5 % and 10 %;
meanwhile, on sample 3 and 4, the bacteria could be
grown at concentration 5 %.
Bidara leaves contain tannin and alkaloid
compounds as antibacterial to inhibit and even kill
bacterial growth. The mechanism of inhibition and
killing of bacterial growth by tannins and alkaloids
that contained in Bidara leaves extract occurs when
the extract solution is inoculated with bacteria and
then incubated at 37° C for 24 hours.
The growth of bacteria at certain concentrations
happened due to the fact that other compounds such
as tannin an alkaloid contained in the ethanol extract
of Bidara leaves could not kill the bacteria; as a
result, the bacteria had become grown. In addition,
at concentrations where the viability of the bacteria
was not found show that tannin and alkaloid could
kill the bacteria Salmonella sp.
Tannin and alkaloid that contained in the extract
of Bidara leaves work together in killing the bacteria
by protein denaturation and inhibit the nucleic acid
bacteria. At first, the alkaline cluster of both of the
active compounds which contain nitrogen will react
with the DNA bacteria, which is the main
component of nucleus. By that process, the protein
synthetic and nucleic acid within cells will be
disrupted so that the metabolism of the bacteria cells
will also be disrupted and the bacteria growth will be
inhibited and even killed (Roslizawaty, et. al., 2013).
Moreover, tannin will also disrupt the wall of the
bacterial cells by creating polysaccharide complex
which can cause the permeability of the bacteria
cells disturbed. The disturbing of bacteria cells
permeability cause the cells cannot be active;
consequently, the growth of bacteria will be
inhibited and also killed (Ustiawaty, et. al., 2015).
The positive control by using ciprofloxacin at
concentration 30 %, 25 %, 20 %, 15 %, 10 %, and 5
% demonstrated that there was no bacteria growth.
It's because ciprofloxacin has the ability to prevent
the forming of gyrase which is the cause of opening
and formation of the superhelix on the DNA
Turbidity of
testing sorvent
The growth of bacteria
Salmonella sp
Chi-Square
36,000
36,000
df
36
36
Asymp. Sig.
,046
,046
ICPS 2018 - 2nd International Conference Postgraduate School
384

bacteria. When the forming enzyme is inhibited, thus
the bacteria cannot be replicated.
Different from the positive control, the negative
control cannot kill the bacteria so that they can grow
because the sterile aquades does not contain any
antibacterial compound to kill the bacteria.
Based on the sample, it is known that there is no
genetic mutation or resistance on the bacteria. This
conclusion refers to the difference of MBC value
from each sample. The value of positive control is 5
%, the MBC of sample 1, 2, and 5 at concentration
15 %, and the MBC of sample 3 and 4 are 10 %. The
other value that also can be understood is that the
ethanol extract of Bidara leaves is unsignificant in
treating the infection caused by Salmonella sp.
4 CONCLUSIONS
Ethanol extract of Bidara leaves can kill the growth
of bacteria Salmonella sp at concentration 30 %, 25
%, 20 %, and 15 %. This finding explicated that the
extract cannot be taken as an alternative treatment
because the bactericidal concentration is under
ciprofloxacin. The MBC sample 1, 2, and 5 are 15 %
while the sample 3 and 4 are 10 %.
The difference of the MBC values indicated that
the profile of Salmonella sp bacteria is different on
the Bidara leaves’ ethanol extract. Furthermore,
from the MBC values, it can be concluded that there
are no bacteria which is resistant or changed
genetically.
ACKNOWLEDGEMENTS
The acknowledgment is conveyed to my academic
mentors who were supervising me in the whole
process of research and in writing the results
findings. Both of my mentors, Dwi Krihariyani and
Nur Cholis Al Anwary, are my undergraduate thesis
supervisor at the Department of Analis Kesehatan,
Poltekkes Kemenkes Surabaya.
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