Download ANTIBIOTIC`S SENSITIVITY IN PATIENT`S DIABETIC FOOT

ANTIBIOTIC’S SENSITIVITY IN PATIENT’S DIABETIC
FOOT ULCER OF Pseudomonas aeruginosa
Pratiwi Apridamayanti1, Khairunnisa Azani Meilinasary1 dan Rafika Sari1
1
Department of Pharmacy, Faculty of Medical, Universitas Tanjungpura, Jalan Prof. Dr. H. Hadari Nawawi, Pontianak
78124, Indonesia
Email : [email protected]
Abstract
Diabetes Mellitus (DM) patients are at risk to have the diabetic ulcer. A main reason for DM’s patient with
ulcer complication to be treated and healed in hospital is caused by bacterial infection. One of many bacteria
that infects diabetic ulcer is Pseudomonas aeruginosa. The effort to treat this infection is by using antibiotic.
The use of antibiotic unfortunately, is often found in accurate causing the microbe resistance to occur. To
choose the right antibiotic, it needs to test the antibiotic’s sensitivity towards Pseudomonas aeruginosa. The
aim of this study is to determine the sensitivity of antibiotics against Pseudomonas aeruginosa. Sample used
was taken from diabetic ulcers swab with grade III and IV Wagner. The identification of bacteria was
managed using the biochemical test and staining Gram test. Antibiotic sensitivity was determined by Kirby
Bauer method. Antibiotic found still sensitive towards Pseudomonas aeruginosa included ciprofloxacin,
norfloxacin, imipenem, levofloxacin, meropenem, ceftriaxone, and cefotaxime. Whereas cefadroxil and
amikacin were resistant towards Pseudomonas aeruginosa in diabetic ulcer grade III and IV Wagner.
Antibiotics that can be used on the bacteria Pseudomonas aeruginosa in diabetic foot ulcer patients are
ciprofloxacin norfloxacin, imipenem, levofloxacin, meropenem, ceftriaxone, and cefotaxime.
Keywords : antibiotic, diabetic ulcer, Pseudomonas aeruginosa
Federation predicts will increase the number
BACKGROUND
of diabetics in Indonesia in 2030 reached 21.3
Diabetes mellitus (DM) is a dangerous
million patients (Wid et al., 2004).
disease that is often called the silent killer.
Patients with diabetes are at risk for
Diabetes mellitus is one of the degenerative
diabetic ulcers. DM patients are estimated to
diseases
handling.
experiencing diabetic ulcers by 15% and 3-
Indonesia is at the fourth position as the
4% exposed to severe infections (Frykberg et
country with the highest number of people
al., 2006). The treatment of diabetic ulcers
with 8.4 million people with the cases of
can be given by reducing the pressure on the
diabetes
skin. Surgery and the use of antibiotics are
that
require
mellitus.
careful
International
Diabetes
deemed important also for the treatment of
streptomycin, ampicillin, and erythromycin.
infected ulcers. Antibiotics are a class of
On the other hand, there are few antibiotics
drugs often used to treat the infection.
that are still sensitive to P. aeruginosa such as
However, the use of antibiotics is often
meropenem, cefotaxime, and amikacin.
imprecise, adverse drug reaction (ADR) and
leads to microbial resistance.
Based on the above background this
research has been conducted to determine the
Diabetic ulcers are open sores on the
sensitivity of antibiotics against Pseudomonas
skin surface. It is possible for complications
aeruginosa bacteria as found in diabetic
makroangiopati
vascular
ulcers Wagner stage III and IV to help direct
insufficiency and neuropathy, the more there
the administration of antibiotics in patients
is a wound on a patient which can develop
with diabetic ulcers.
causing
into an infection caused by aerobic and
anaerobic bacteria. (Tambunan, 2007). In
Objectives
patients with ulcers of diabetic Gram-negative
The aim of this study is to determine the
bacteria are identified at most, which is 7
antibiotic sensitivity class of aminoglycoside
times more compared with gram-positive
(amikacin), class of cephalosporin (cefadroxil,
bacteria (Aulia, 2008).
ceftriaxone,
Based
on
research
Sari
and
the
gram-negative
levofloxacin
such
as
cefotaxime),
class
of
carbapenem (imepenem and meropenem), and
Apridamayanti (2015) it has been found that
bacterium,
and
class
of
quinolones
and
(ciprofloxacin,
norfloxacin)
against
Pseudomonas aeruginosa is one of the
Pseudomonas aeruginosa at the foot ulcers of
bacteria that have the highest percentage in
diabetic degrees III and IV Wagner. It also
patients with diabetic ulcers. It is also
included the resources for the promotion of
reinforced in research Manisha (2012) that the
the prevention of antibiotic resistance on
Gram-negative bacterial pathogens in diabetic
patients with diabetic ulcers degrees III and
ulcers mostly is Pseudomonas aeruginosa 48
IV.
(30.57)%,
Klebsiella
spp
35
(22.29),
Escherichia coli 26 (16.56%) and Proteus sp 8
METHOD
(4.37%).
As revealed in the research of Aulia
Sampling
(2008) Pseudomonas aeruginosa has the
Samples of bacteria were taken from diabetic
highest levels of resistance to doxycycline,
ulcer swab degrees III and IV Wagner using a
sterile swab, then stored in a sterile transport
Clinical Laboratories and Standards Institute
medium and sealed. The number of ethical
(2014).
clearence is 4270/ UN 22.9/ DT/ 2015.
Bacteria contained in a sterile swab planted in
RESULT AND DISCUSSION
the media blood agar and Mac-Conkey.
Isolation of Pseudomonas aeruginosa
The bacteria grown in two media
Isolation of Pseudomonas aeruginosa
The bacteria are grown on media blood agar
included Mac-Conkey Agar (MCA) and
and Mac-Conkey. Planting bacteria were
Blood Agar Plate (BAP). This planting used
performed directly on solid agar media and
the scratch method that has been selected for
incubated for 24 hours in an incubator at a
being more practical, economical and not time
temperature of 32-40 °C (Aulia, 2008).
consuming as compared to the casting method
Identification of Pseudomonas aeruginosa
Identification has been performed with the
Gram stain test and biochemical tests.
Biochemical test was conducted on the test
fermentation of sugars, fermentation of
carbohydrates,
motility,
indole,
H2S
production, urea, oxidase, and fermentativeoxidative.
method used antibiotic disks. Media used was
Mueller Hinton Agar (MHA). Meanwhile, the
antibiotic disk used included ciprofloxacin,
imepenem
lubricated on both media, and then looped
round heated to glow aside some time after
being etched in a zigzag pattern on both
media. Planting bacteria were carried on solid
agar medium and incubated for 24 hours in an
incubator at a temperature of 32-40 oC. Once
bacteria grew on both media.
Tests performed using the Kirby-Bauer
cefadroxil,
First of all, a sterile swab in amies media was
morphological observation was completed
Antibiotic Sensitivity Testing
norfloxacin,
requiring longer time and more materials.
levofloxacin,
ceftriaxone,
and
amikacin,
cefotaxime,
meropenem.
The
determination of antibiotic sensitivity was
performed based upon the guidelines of
Mac Conkey (MC) is a selective
differential medium used to see the ability of
bacteria
to
ferment
glucose.
P.aeruginosa colonies when grown will not
be coloured because it is not able to ferment
lactose as shown in Figure 1. The blood agar
media is a differential medium that can
differentiate bacteria based on their ability to
lyse red blood cells. P.aeruginosa colonies
formed are round, convex, transparent and
uneven
edges. P.aeruginosa
experience
replaced by safranin red. Safranin got into the
haemolysis Beta (β) or so-called haemolysis
cells of bacteria and replaced crystal violet so
total, defined as the entire lysis of red blood
that the colours seen are red.
cells. A clear zone, close to the colour and
Bacterial biochemical test is a method
transparency of a basic media, surrounded the
or treatment performed to identify and
colony as seen in Figure 2.
determine a pure culture of the isolated
bacteria through the properties of physiology.
Identification of Pseudomonas aeruginosa
Table 1 shows that the bacteria Pseudomonas
The identification of Pseudomonas
aeruginosa in motility test was positive in this
aeruginosa was performed using Gram
case as shown by the spread of white as the
staining and biochemical tests. Biochemical
roots around the inoculation. This showed the
tests included the fermentation of sugars,
movement of bacteria inoculated, indicating
fermentation
motility,
that these bacteria had flagella. The bacteria
indole, H2S production, urea, oxidase, and
were also positive in the test Simmon citrate
fermentative-oxidative.
as indicated by a colour change from green to
of
carbohydrates,
As seen in figure 3, Gram staining test
results
showed
that
the
bacterium
blue.It shows that these bacteria utilize citrate
as a carbon source.
P.aeruginosa was a Gram-negative bacterium
Oxidase test is a biochemical reaction
for showing some results in red. The results
carried out to see their cytochrome oxidase,
were obtained in accordance with the
an enzyme that is usually called indophenol
description
stating
oxidase. Test oxidative/fermentative was
that P.aeruginosa has been shaped gram-
conducted with an aim to know the nature of
negative
oxidation or fermentation of bacteria to
Mayasari
bacterium
aeruginosa is
a
(2006)
bacillus, Pseudomonas
Gram-negative
bacteria
glucose by using two tubes, one of which was
because bacteria are in red cells. The red
as
media to
use paraffin. Pseudomonas
colour as seen in bacterial cells was due to the
aeruginosa results of discoloration on one of
release of the dye crystal violet for leaching
the tubes from green to yellow indicate the
using alcohol. The cell wall of Gram-negative
bacteria from oxidative.
bacteria that have a peptidoglycan layer is
The results showed that the bacterium
thinner than Gram-positive bacteria. Gram-
was identified as the bacterium Pseudomonas
negative bacteria with a thin peptidoglycan
aeruginosa. These results are consistent with
layer would be more easily dislodged and
Sulistiyaningsih (2010) showing that the
bacteria
Pseudomonas
biochemical
tests
aeruginosa in
of
glucose,
be ongoing. This separation will always result
lactose,
in the excessive twisting double helix of DNA
mannitol, maltose, sucrose, indole, urea,
prior to the point of separation. This
H 2 S showed a negative result on the test,
mechanical barriers can be overcome with the
while motile, oxidase and Simmons citrate
help
showed positive ones.
(topoisomerase II). Whereas levofloxacin
of bacteria DNA gyrase
enzyme
antibiotics work by inhibiting topoisomerase
II and IV in bacteria. Topoisomerase II
Antibiotic Sensitivity Test
Antibiotic
was
enzyme function causing relaxation of the
samples
DNA strands are experiencing excessive at
Pseudomonas aeruginosa and was selected
the time of transcription in the DNA
randomly. Media test using Mueller Hinton
replication
Agar (MHA). The disk shaped antibiotics
function in the separation of the newly formed
were used so that unnecessary antibiotic
DNA after bacterial DNA replication process
suspensions
is complete (Setiabudy, 2011).
carried
out
sensitivity
on
the
testing
positive
manufacture
antibiotics.
Antibiotics were tested i.e. cefotaxime,
ceftriaxone,
norfloxacin,
meropenem,
levofloxacin,
imepenem,
amikacin,
cefadroxil, and ciprofloxacin.
process.
Topoisomerase
IV
Amikacin is an aminoglycoside class
of
antibiotics. The
mechanism
of
aminoglycosides, is when aminoglycosides
have been entered into the bacterial cell, and
Based on the test results of sensitivity
the aminoglycosides bind to the 30S ribosome
as seen in Table 2, the sensitive antibiotics
and inhibit protein synthesis. The binding of
included
ciprofloxacin,
norfloxacin,
aminoglycosides in this ribosome accelerates
meropenem,
levofloxacin,
the aminoglycoside transport into the cell,
ceftriaxone and cefotaxime while cefadroxil
followed by a cytoplasmic membrane damage
and amikacin-resistant bacteria P. aeruginosa.
and
imipenem,
Ciprofloxacin,
norfloxacin
subsequent
cell
death. It
occured
and
allegedly and is a genetic mutation that results
levofloxacin are the quinolone class of
in the disruption of protein synthesis. In this
antibiotics. The mechanisme of ciprofloxacin
case, the wrong types of amino acids in a
and norfloxacin was by inhibiting the enzyme
polypeptide chain spliced to form a type of
DNA gyrase in bacteria. Double helix shape
protein that is wrong. While the decline in the
of DNA must be separated into two chains of
antimicrobial activity of amikacin was caused
DNA during replication and transcription will
by the modification of enzymes, efflux pumps
and increased activity as occurred 16S rRNA
also bind to beta-lactam antibiotics causing
methylation. Modification enzymes that occur
this enzyme not able to catalyse the cell wall
can
enzyme
transpeptidation although still continuing to
by
be formed. The cell walls that form does not
nukleotidiltranferase and phosphorylation by
have a crosslinking and peptidoglycan formed
fosforiltranferase. Increased efflux pumps
is not perfect making it weaker and easily
may occur because of the XY gene Mex -
degraded. In
OPR M which encodes the activation of
difference in osmotic pressure within the
efflux pump. Methylation 16S rRNA gene can
cell Pseudomonas aeruginosa bacteria in the
occur because RmtA, RmtB, ARMA and
environment and will make the occurrence of
RmtD which encodes bacteria (Setiabudy,
cell lysis. In addition, the complex protein
2011).
transpeptidase and beta-lactam antibiotic will
be
acetylated
acetyltransferase,
by
the
adenylation
Cefotaxime and ceftriaxone are third
generation
cephalosporin
antibiotics. As
with
class
other
of
beta-lactam
a
normal
condition,
the
stimulate autolysin compounds that can
mendigesti
cell
walls
bacteria Pseudomonas
aeruginosa,
of
and
antibiotics, the mechanism of action is a
then Pseudomonas aeruginosa lose cell wall
cephalosporin antibiotic inhibits bacterial cell
or through lysis will die (Setiabudy, 2011).
wall synthesis, which inhibited the reaction
Cefadroxil
is
a
first
generation
trans-peptidase is the third stage in the
cephalosporin class of antibiotics. This class
reaction
of antibiotics is more effective for Gram-
sequence
cell
wall
formation
(Setiabudy, 2011).
positive bacteria. Cefadroxil resistance is
Imipenem and meropenem are a class
caused by the formation of beta-lactamase
of carbapenem antibiotic as the large class of
enzymes. This enzyme is produced from gene
beta-lactamase. Beta-lactamase
antibiotics
TEM1, TEM2, and SHV1 and can inhibit the
work to kill the bacteria in a way of inhibiting
action of cefadroxil namely by hydrolysing
the cell wall synthesis. In the process of the
the beta-lactam ring contained in cefadroxil so
formation of cell walls, transpeptidation
that this drug can not bind to its receptor
reaction was catalysed by the enzyme
(Meletis and Bagkeri, 2013).
transpeptidase and produced a crosslinking
between
two
Transpeptidase
peptide
enzyme
chains-glucan.
located
on
the
cytoplasmic membrane of the bacteria can
CONCLUSION
Pseudomonas
aeruginosa
isolates
were sensitive to the antibiotic levofloxacin,
norfloxacin,
cefotaxime,
Development and Treatment Options.
Greece: Licensee Intech.
ceftriaxone,
Mayasari E. (2006) Pseudomonas aeruginosa:
ciprofloxacin, imipenem, meropenem while
Karakteristik, Infeksi dan Penanganan.
the bacteria Pseudomonas aeruginosa are
Medan: USU Repository.
resistant to antibiotic cefadroxil and amikacin.
PERKENI. (2011). Konsensus Pengelolaan
dan
Acknowledgments
Pencegahan Diabetes Mellitus
Tipe 2 di Indonesia. Jakarta : FKUI
The authors are thankful to DIPA
Tanjungpura University for research about the
RSCM.
Sari R, Apridamayanti P. (2015) Identifikasi
Identification of Bacteria ESBL Producing
Bakteri
Diabetic Ulcer in Grade III and IV Wagner.
Penderita Ulkus Diabetikum Derajat
Penghasil
ESBL
pada
III dan IV Wagner. Pontianak :
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