Expression of Caspase-3 in the Liver and Spleen Rattus norvegicus

Which Infects Bacteria Klebsiella pneumonia and Klebsiella

pneumoniae Extended Spectrum Beta Lactamase

I Gede Andika Sukarya

, Willy Sandhika.

and Agung Dwi Wahyu Widodo

Department of Immunology Postgraduate School Universitas Airlangga, Surabaya, East Java, Indonesia

Department of Anatomy Pathology, Faculty of Medicine, Universitas Airlangga, Surabaya, East Java, Indonesia

Department of Microbiology Clinic, Faculty of Medicine, Dr. Soetomo Hospital, Indonesia

Keywords: Apoptosis, Caspase-3, Klebsiella pneumoniae, Klebsiella pneumoniae ESBL.

Abstract: Klebsiella pneumoniae (K. pneumoniae) is found in nosocomial infections and Gram-negative number two

is the most dangerous. The bacteria K. pneumoniae causes unusual nosocomial infections in hospital

settings. Drug resistance in bacteria K. pneumoniae ESBL production may increase the risk of death at the

time of treatment. Caspase-3 is the hallmark of apoptosis. It can be used as an indicator of virulence of the

infection of K. pneumoniae. This research aims to look at the effects of infection of K. pneumoniae and K.

pneumoniae ESBL against eksprsi caspase-3 in the liver and spleen of rats Rattus novergicus. The study

uses 12 healthy rats that were divided into three groups, namely the control group, infection K. pneumoniae,

and infection K. pneumoniae ESBL. Each group of samples taken from the liver and spleen organs after 24

hours of the infection process and immunohistochemical expression to see caspase-3. Of the research results

obtained, the percentage of caspase-3 expression in the liver and spleen organs in rats that infect bacteria K.

pneumoniae, are higher than K. penumoniae ESBL. The expression of caspase-3 in the liver and spleen

organs is steeper than the infection of K. pneumoniae. We conclude that the bacteria K. pneumoniae is more

virulent than K. pneumoniae ESBL.

1 INTRODUCTION

Klebsiella pneumoniae (K. pneumoniae) is found

in nosocomial infections and Gram-negative number

two is the most dangerous (Tsai et al., 2009). The

bacteria K. pneumoniae causes unusual nosocomial

infections in hospitals (Woldu, 2016). The bacteria

K. pneumoniae is very dangerous for patients with

immunocompromised and immunodeficiency,

causing damage to organs (Wu, 2015). The

transmission of K. pneumoniae in hospitals has

become a particular concern in Europe, the United

States, Argentina, and Australia. Infections caused

by K. pneumoniae result in longer treatment times in

hospital and resistance to antibiotics (Brisse et al.,

2006).

Extended Spectrum Beta Lactamase (ESBL) K.

pneumoniae produces an aggravating risk factor for

infection. The bacteria K. pneumoniae ESBL

production is resistant to beta-lactam antibiotics and

are at risk of adding to long treatment times in ICUs

(Toner et al., 2016). The virulence of K. pneumoniae

this decade can infect normal or healthy individuals,

due to resistance to drugs and hipervirulen (Paczosa,

2016)

Apoptosis plays a role in bacterial infection.

Apoptosis affects the immune cells that are very

important in the course of the infection. Regulation

of apoptosis is an important aspect of the host cell

response to stress and infection and should be

continual. Caspase in the apoptosis process plays a

role in cellular processes, which occur before

apoptosis happens (Silva, 2009). Apoptosis triggered

by the TNFα is produced by inflammation from the

bacterial infection Klebsiella pneumuniae good

through the intrinsic or extrinsic pathways triggered

by various cellular responses through the endotoxin

lipopolysaccharide (LPS). The Endotoxin bacteria K.

pneumuniae often triggers endothel cell damage so

multiple organ failure can occurs. Endotoxin triggers

the inflammatory process, which is sustainable and

increases the incidence of cell death in the follicle

cell dendritic, dendrite cells, neutrophils, CD4

244

Andika Sukarya, I., Sandhika, W. and Wahyu Widodo, A.

Expression of Caspase-3 in the Liver and Spleen Rattus norvegicus Which Infects Bacteria Klebsiella pneumonia and Klebsiella pneumoniae Extended Spectrum Beta Lactamase.

DOI: 10.5220/0007540702440248

In Proceedings of the 2nd International Conference Postgraduate School (ICPS 2018), pages 244-248

ISBN: 978-989-758-348-3

CD8

, and B cells (Paczosa, 2016). Caspase-3 is a

death protease activation of mediators that

frequently programs cell death (apoptosis), and

catalyzes the cleavage of cell specific proteins of

some. Caspase-3 is the hallmark of apoptosis, and

indispensable for the condensation of the cell

apoptosis and fragmentation of DNA. Caspase-3 is

very important in the process of apoptosis (Porter,

1999).

The spleen immune system is responsible for

protecting the body from the invasion of pathogens

and detecting old cells, damaged mechanically, and

distorted, which can lead to the formation of tumors.

The spleen plays out a double simultaneous reaction

against bacterial antigens and allogenes. As a

secondary lymphatic system, due to the number of

lymphocytes in the spleen area, more and bacteria

will be taken towards the organ spleen.

Lymphocytes in the spleen undergo apoptosis. The

liver is the largest location of kupffer cells, NK cells,

and NKT cells. Cells, NK cells and kupffer NKT

cells are activated by the stimulation of APC, both

directly and indirectly, to respond to bacterial

infections. Activation of the cells that are in the liver

apoptosis are triggered to respond to infections

(Wang, 2014). The expression of caspase-3 denotes

prosen apoptosis, which occurs during the ongoing

infection process (Porter, 1999).

In this study, we will present the expression of

caspase-3 in infection K. pneumuniae and K.

pneumuniae production of ESBL in Rattus

norvegicus. Expression of caspase-3 in the liver and

spleen of a rat show virulence infection K.

pneumuniae and K. pneumuniae ESBL. This can be

seen with the virulence of the infection K.

pneumuniae and K. pneumuniae in the liver and

spleen organs.

2 MATERIALS AND METHODS

2.1 Animals

The rat Rattus novergicus; male rats that are healthy

and not exposed to infection. They are characterized

by the movement of agile rats, aged three months,

with a weight of 200–250 grams.

2.2 Bacteria

The bacteria K. pneumoniae ESBL and K.

pneumoniae derived from Installations Clinical

Microbiology of Dr. Sutomo Surabaya. Made with

CFU of bacteria concentrations of Phosphate-

buffered saline (PBS).

2.3 Method

The healthy rat is selected by raffled and is given a

mark or code of any group consisting of four rats.

Injection materials on the peritoneum in Quadrant 3

included as much as 1ml (Group control in injection

PZ (Phisiological zouth), a group of K. penumoniae

injection on concentrations with 10

CFU K.

pneumoniae, and groups of K. pneumonie ESBL

injection on concentrations with 10

CFU K.

penumonie ESBL) in each group of animals. The

rats were observed for 24 hours. The livers and

spleen organs were retrieved 24 hours after surgery.

The rat’s liver and spleen fixation buffer into the

tissue preparation and formaldehyde (formalin fixed

and paraffin embedded section). The sample was

made of paraffin blocks and cutting samples with a

thickness of 4µm, using either a microtome or tool

placed on the microscope slide. An immunostaining

process was performed using the

immunohistochemistry reagents Caspase-3 (Bioss

Antibodies, Bioss, USA).

2.4 Immunohistochemistry

Deparaffinization and rehydration; Wash slides

twice in Xylene for three minutes each time in real

time; Wash slides in Xylene 1:1 with 100% ethanol

for three minutes in real time; Wash slides twice in

100% ethanol for three minutes each in real time;

Wash slides twice in 95% ethanol for three minutes

each in real time; Wash slides in 70% ethanol for

three minutes in real time; Wash slides in 50%

ethanol for three minutes in real time; Rinse slides

gently with running distilled water for five minutes

in real time. Antigen retrieval; Boil slides in 0.01M

sodium citrate buffer (pH6) at 100°C for 15–20

minutes; Remove the slides from the heat and allow

them to stand at real time in the buffer for 20

minutes; Rinse twice with TBST for five minutes in

real time. Immunostaining; block with endogenous

peroxidase with 3% hydrogen peroxide for 30

minutes; block with 5% serum or BSA for two hours

in real time; drain blocking buffer from slide;

incubate slides with the diluted primary antibody

overnight at 4°C with gentle agitation; Wash slides

twice with TBST for five minutes in real time;

incubate slides with diluted conjugated secondary

antibody for two hours in real time with gentle

agitation; wash slides twice with TBST for five

minutes in real time; develop with chromogen for 10

Expression of Caspase-3 in the Liver and Spleen Rattus norvegicus Which Infects Bacteria Klebsiella pneumonia and Klebsiella

pneumoniae Extended Spectrum Beta Lactamase

245

minutes in real time; wash slides in distilled water

for one minute in real time; counterstain (if

required); Dehydrate when using a chromogen

substrate that is alcohol insoluble by washing slides

in 80%, 95%, 100%, and Xylene each for one

minute in real time; Mount coverslips; check out the

slides and calculate the percentage of positive cells

caspase-3 there are liver and lymphatic organs.

3 RESULT

The research results were obtained as a percentage

of the number of cells that undergo caspase-3 on the

organ spleen and liver in the treatment group

infection K. pneumoniae, K. pneumoniae ESBL and

the control group (Figures 1 and 2).

Figure 1: Box-plots the percentage of cells that undergo

caspase-3 in the spleen.

Figure 2: Box-plots the percentage of cells that undergo

caspase-3 in the liver.

Figure 1 shows the percentage of caspase-3 in

the lymphatic organs. The group treatment in

infection K. pneumoniae are higher than group

treatment on infection of K. pneumoniae ESBL and

those in the control group. The value of the median

is the highest to the lowest in succession, i.e. the

Group of K. pneumoniae (45.5), K. pneumonia

ESBL (35), and the control group (4).

Figure 2 shows the percentage of caspase-3 in

the liver organ on group infection K. pneumoniae in

treatment was higher in the treatment group of

infection K. pneumoniae ESBL and those in the

control group. The value of the median is the highest

to lowest in succession i.e., the Group of K.

pneumoniae (81), K. pneumonia ESBL (60.5) and

the control group (9).

Figure 3 shows R. norvegicus spleen cells

demonstrating the immunostaining Caspase-3; The

control group is shown in picture A with a

magnification microscope 1000x; K. pneumoniae

group is shown in picture B with a magnification

microscope 1000x. There are many cells that express

caspase-3, such as arrows. Labelled cells in the

spleen are experiencing the process of apoptosis, and

groups of K. pneumonia ESBL in picture C shows

the cells at 1000x magnification. On the organ in a

bacterial infection K. pneumoniae and K.

pneumoniae ESBL shows the expression of caspase-

3 on the cell. This marks the process of apoptosis

occurring on the spleen, caused by a bacterial

infection K. pneumoniae, causing almost 60% of

damage to the spleen organ.

Figure 3: Cells in the spleen tissues of R. norvegicus in

immunostaining caspase-3 in the control group Figure A,

K. pneumoniae Group Figure B and K. pneumonia ESBL

Figure C.

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Figure 4: Cells on R. norvegicus in liver tissue

immunostaining caspase-3 in the control group Figure A, ,

K. pneumoniae group Figure B and K. pneumoniae ESBL

group Figure C.

Figure 4 shows R. norvegicus demonstrating the

liver tissue in immunostaining caspase-3; the control

group is shown in picture A at 1000x magnification

using a microscope; the K. pneumoniae group is

shown in picture B at 1000x magnification using a

microscope. There are many cells that express

caspase-3, such as arrows and labelled cells in the

liver that are experiencing the process of apoptosis.

For the K. pneumonia ESBL group, picture C shows

cells at 1000x magnification using a microscope.

On the organ in a bacterial infection, K. pneumoniae

and K. pneumoniae ESBL shows the expression of

caspase-3 on the cell. This marks the process of

apoptosis occurring in liver organs caused by the

bacterial infection K. pneumoniae, causing 80%

liver organ damage.

4 DISCUSSION

There was an increase in the percentage of caspase-3

in liver and spleen organs in a group of animal

models with the infections K. pneumoniae and K.

pneumoniae ESBL. Injection of germs in an animal

model is done through the peritoneal line. The test

compound injected into the peritoneal cavity will be

absorbed into the portal circulation and transported

to the liver (Shayne et al., 2013). The liver receives

the blood vein of the portal and the arterial blood,

liver, and spleen are important components in the

defense against infection entering the blood stream.

To achieve this role, the liver and the spleen contain

many innate and adaptive immune cells specifically

to detect and capture pathogens from the blood

stream. Furthermore, the immune cells participate in

the immune response that leads to the purge of

pathogens, the recruitment of leukocytes and antigen

presentation to lymphocytes in the blood stream. An

increasing number of caspase-3 in liver organs can

be caused due to a high inflammatory process, which

triggers the cells to hepatocyte or innate underwent

apoptosis. There is a balance between activation and

tolerance that characterizes the liver and spleen as

immunological organ front line (Jenne and Kubes,

2013).

The spleen is an organ of the lymphatic system

and inserted into the bloodstream is a collection of

lymphoid tissues. The spleen immune system is

responsible for protecting the body from the

invasion of pathogens and detecting old cells,

damaged mechanically and distorted that can lead to

the formation of tumors. Recent studies prove the

dominant role in the simultaneous double reaction

against bacterial antigens and allergens. The spleen

is the seat of an innate and adaptive immune system.

Microbial network penetration evokes an innate

system direct reaction, while the adaptive immune

response involves the interaction of cells that

recognize specific antigens in the context with the

MHC presented by a cell that gives rise to antigens

(Wluka et al., 2006) A secondary lymphatic system

allows the number of lymphocytes in the spleen area

to increase, and infected bacteria is carried towards

the spleen organ. Lymphocytes in a lien allows will

undergo apoptosis. Of the function that has been

described above, an increasing number of caspase-3

in liver and spleen organ can be caused due to the

high inflammatory process to trigger the cells to

hepatocyte or undergoing innate apoptosis.

Caspase-3 positive cells in the spleen and liver

organs in animal models with the infection of K.

pneumoniae are higher than animal models in

infection of K. pneumoniae ESBL. The bacterium K.

pneumoniae and K. pneumonie ESBL triggered a

wide range of cellular response through the

endotoxin lipopolysaccharide (LPS). The ongoing

process of inflammation triggers endotoxins and

increases the incidence of apoptosis on dendrite

cells, follicular dendritic cells, and neutrophils

nanotechnologies, the number of CD4

, CD8

and B

cells (Paczosa, 2016). K. pneumoniae protects a

variety of humoral defense mechanisms, such as the

mechanism of bacterial resistance to complement the

damage. In phagocytosis, this did not happen on K.

pneumoniae ESBL. Human beta-defensins 1 (HBD-

1) and HBD-2 inefficient kill K. pneumoniae as

HBD-3. K. pneumoniae ESBL production is more

susceptible to HBD (Moranta et al., 2010). Caspase-

3 indicates that high apoptosis occurrence is very

high and is prominent regarding pathological liver

Expression of Caspase-3 in the Liver and Spleen Rattus norvegicus Which Infects Bacteria Klebsiella pneumonia and Klebsiella

pneumoniae Extended Spectrum Beta Lactamase

247

disease. The end result of caspase-3 is the high cause

of dysfunction of the liver, cirrhosis, and

tumorigenesis (Wang and Lien, 2013).

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