Differences of Caspase-3 Expression in the Spleen and Liver of Sepsis

Models in Rats Infected with Escherichia coli ESBL and Klebsiella

pneumoniae Carbapenemase

Lisa Savitri

, Willy Sandhika

, and Agung Dwi Wahyu Widodo

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

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

Department of Microbiology Clinic, Faculty of Medicine, RSUD Dr. Soetomo, Surabaya, East Java, Indonesia

Keywords : Caspase-3 expression, Escherichia coli ESBL, Klebsiella pneumoniae carbapenemase

Abstract : Sepsis is the leading cause of death in the world. Sepsis patients with Extended Spectrum β-lactamase

(ESBL)-producing bacterial infections were 57.4% Escherichia coli, 21.35% Enterobacter sp, and 21.3%

Klebsiella sp. Caspase-3 is the most important caspase effector responsible for morphological and biological

changes in apoptotic cells. This type of research is true experimental with a post-test only control group

design, using one control rat group and two groups of rats infected with E. coli Extended Spectrum β-

lactamase (ESBL) and Klebsiela pneumoniae carbapenemase (KPC) for 24 hours to find out the different

expression of caspase-3 in the spleen and liver of those infected rats. Expression of caspase-3 was observed

by staining the spleen and liver with caspase-3 p12 subunit antibody. Cells expressing caspase-3 were

counted under the light microscope. The results showed that caspase-3 expression in the KPC infected

spleen group was 65.25±12.69%, whereas E. coli ESBL was 33.75±3.862%. This is thought to be

influenced by the presence of antigen differences between the two bacteria, thus the possibility of apoptosis

in lymphocyte cells caused by KPC would be higher when compared with those infected with E. coli ESBL.

Caspase-3 liver expression in the KPC group had a value of 58.75±4.031%, while the E. coli ESBL infected

was 48.75±6.292%. It may be affected by differences in soluble factors of both bacteria, thus the possibility

of apoptosis in hepatocyte-induced cells by KPC will be higher when compared with those that are E. coli

ESBL infected.

1 INTRODUCTION

Sepsis is a clinical syndrome that occurs due to

excessive body response to stimulation of

microorganism products (Guntur, 2007). Sepsis is

the leading cause of death in the world and the cause

of deaths in Intensive Care Units (ICU). It is

estimated that about 1,400 patients die in the ICU

because of sepsis (Poeze et al., 2004). Apoptosis is

commonly involved in bacterial infections and

pathogenesis. During bacterial infections, virulent

factors (mostly endotoxins) are produced and

secreted from pathogens and trigger apoptotic

signals. Research on caspase-3 is important, as it is

the most important caspase effect responsible for

morphology and biological changes seen in

apoptotic cells (Ghatage et al., 2013). Based on this

phenomenon, it is necessary to conduct research to

determine the increase of expression caspase-3 on

the spleens and livers of rats infected with

Escherichia coli Extended Spectrum β-lactamase

(ESBL) and Klebsiela pneumoniae carbapenemase

(KPC).

2 METHODS

2.1 Type and Design of Research

The type of this research is pure laboratory (true

experimental) research using post-test only for the

control group (data retrieval done after treatment)

and compared with the control group.

Savitri, L., Sandhika, W. and Wahyu Widodo, A.

Differences of Caspase-3 Expression in the Spleen and Liver of Sepsis Models in Rats Infected with Escherichia coli ESBL and Klebsiella pneumoniae Carbapenemase.

DOI: 10.5220/0007540802490252

In Proceedings of the 2nd International Conference Postgraduate School (ICPS 2018), pages 249-252

ISBN: 978-989-758-348-3

249

2.2 Place and Time of Research

2.2.1 Research Place

This study was conducted in several locations: the

Animal Unit Laboratory of Biochemistry, Faculty of

Medicine, Universitas Airlangga, Microbiology

Laboratory of RSUD Dr. Soetomo, Surabaya, and

Anatomical Pathology Laboratory, Faculty of

Medicine, Universitas Airlangga.

2.2.2 Research Time

This study was conducted for approximately three

months, from October 2017 to December 2017.

2.3 Research Objects

The object of the research used in this research was

rats (Rattus norvegicus); a male strain Wistar aged

about eight to 12 weeks with a body weight of 150–

200 grams that came from the animal unit’s

biochemistry laboratory, Faculty of Medicine,

Universitas Airlangga.

2.4 Animal Treatment

Adapted rats were injected in the peritoneum section

with the following treatments: 1) group one as

normal control, i.e. injected aqua pro-injection-free

pyrogen; 2) group two as treatment one, i.e. injected

rat E. coli ESBL with dose 1x10

CFU/ml; and 3)

group three as treatment two, i.e. rat injected KPC

with dose 1x10

CFU/ml. After 24 hours post-

exposure of polymicrobial sepsis animals will show

apoptosis in the spleen and liver.

2.4.1 Caspase-3 Expression Observation on

the Spleen and Liver of Rats

Observations of the caspase-3 expression on the

liver and spleen of rats were performed by painting a

primary antibody caspase-3 p12 subunit antibody

(host: rabbit, target protein: caspase-3 p12 subunit,

clonality: polyclonal, isotype: IgG, entrez gene: 836,

source: KLH conjugated synthetic peptide derived

from human caspase-3 p12 subunit, purification:

purified by protein A) Bioss Antibodies production.

The caspase-3 expression was observed using the

immunohistochemical method. Caspase-3 expressed

when exposed to the brown color in cytoplasmic

sections, but if in clear cytoplasmic sections, it can

be stated that caspase-3 is unexpressed. The

calculation of caspase-3 expression is done by

calculating the cell expressing caspase-3 divided by

all the preserved cells, then multiplying by 100%, so

the data is expressed using a percentage.

3 RESULT

3.1 Caspase-3 Expression in Spleen of

Rats with E. coli ESBL and KPC

Table 1 : Average Data and Standard Deviation Caspase-3

Expression in a Rat’s Spleen with Control Treatment, E.

coli ESBL Infection, and KPC Infection (%).

Group x±SD Median Max-Min

Control

(n=4)

4,25±0,5 4 5–4

E. coli

ESBL

(

n=4

)

33,75±3,86

33,5 38–30

KPC (n=4)

65,25±12,6

67 78–49

Figure 1 : Expression of caspase-3 in spleen of rats

infected with E. coli ESBL are stained brown (green

arrow). Magnification: x1000.

ICPS 2018 - 2nd International Conference Postgraduate School

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Figure 2 : Expression of caspase3 in spleen of rats

infected with KPC are stained brown (green arrow).

Magnification: x1000.

3.2 Caspase-3 Expression in the Livers

of Rats with E. coli ESBL and KPC

Table 3.2 Average Data and Standard Deviation Caspase

3 Expression on Rat’s Liver with Control Treatment, E.

coli ESBL Infection, and KPC Infection (%).

Group x±SD Median Max-Min

Control

(n=4)

9,5±2,38 9,5 12–7

E. coli

ESBL

(n=4)

48,75±6,29

46,5 58–44

KPC (n=4)

58,75±4,03

59 63–54

Figure 3 : Expression of caspase-3 in livers of rats

infected with Escherichia coli ESBL are stained brown

(green arrow). Magnification: x1000.

Figure 4: Expression of caspase-3 in the livers of rats

infected with K. pneumoniae carbapenemase are stained

brown (green arrow). Magnification: x1000.

4 DISCUSSION

4.1 Caspase-3 Expression in the Spleen

of Rats with E. coli ESBL and KPC

Increased caspase-3 expression in rat-infected KPC

was higher when compared with those infected with

E. coli ESBL. It can be influenced because of

differences in antigens possessed by KPC with E.

coli ESBL. The KPC capsule consists of an O

antigen which is a liposaccharide consisting of a

repeating polysaccharide unit. Antigen O is resistant

to heat and alcohol. The second antigen is the K

antigen. The K antigen is outside the antigen O and

is a capsular polysacharida. The K antigen may

interfere with agglutination through antiserum O and

is associated with virulence. Both of these antigens

increase the pathogenity of KPC.

During bacterial infections, virulent factors are

produced and secreted by pathogens and trigger

apoptotic signals. In general, cells undergo apoptosis

of two main pathways, extrinsic pathways (dead

receptor pathways) and intrinsic pathways

(mitochondrial pathways) (Jin and El-Deiry, 2005;

Ayala et al., 2007). After releasing specific pro-

apoptotic proteins, such as cytochrome c,

smac/DIABLO, AIF, and Endo G, the execution

path begins with caspase activation 3. Its main

purpose is to bind and activate the caspase

recruitment domain (CARD), Apaf-1, and procaspse

9, which leads to the formation of apoptosome.

Next, it leads to activation of caspase-9 and further

Differences of Caspase-3 Expression in the Spleen and Liver of Sepsis Models in Rats Infected with Escherichia coli ESBL and Klebsiella

pneumoniae Carbapenemase

251

activates the caspase-3 effector, which completes the

apoptotic pathway. These are apoptosome

formations and activation of the caspase effectors

that cause apoptotic events, such as chromatin

condensation, plasma membrane asymmetry, and

cellular blebbing (Abud, 2004; Nikitakis et al.,

2004).

4.2 Caspase 3 Expression in the Livers

of Rats with E. coli ESBL and KPC

Based on previous research, it was found that there

was an increase of caspase-3 expression in the livers

of the of rats in the group infected with E. coli ESBL

and infected by KPC. Injection of bacteria in the rats

was done through an intraperitone injection

pathway. Bacteria injected into the peritoneum

cavity will be absorbed into the portal circulation

and transported to the liver. As an organ that acts as

a recipient of portal and arterial blood vessels, the

liver is an important component in the defense

against blood-borne infections.

Increased caspase-3 expression in rats

infected with KPC was higher when compared with

E. coli ESBL-infected rats. This might be affected

by differences in soluble factors of bacteria that can

induce host cells. Factors involved in the virulence

of KPC strains include capsular serotypes,

lipopolysaccharides, ironscavenging systems,

fimbrial and non-fimbrial adhesions. The

polysaccharide capsule surrounding KPC protects

itself against the action of phagocytosis and serum

bactericidal and may be considered the most

important determinant of the virulence of KPC.

Liver damage is associated with the incidence of

liver cell apoptosis (Mordue et al., 2001). Apoptosis

through intrinsic pathways in liver cells is caused by

a soluble factor of bacteria that can induce host cells,

and is thus toxic to other cells. This soluble factor

causes the mitochondria to release ROS. These

bacterial infections cause the mitochondria to

produce ROS and trigger the release of cytochrome

c (Nomura et al., 2000). Cytochrome c will trigger

caspase-9 to bind to the caspase effect, i.e. caspase-

3, resulting in apoptosis (Yoon et al., 2002).

5 CONCLUSIONS

The increase in the caspase-3 expression in the

spleens of rats infected with KPC was higher

compared with that of rats infected with E. coli

ESBL. The different antigens in those two different

bacteria may have contributed to the expression of

caspase-3 and the possibility of apoptosis in the

lymphocyte cells caused by KPC would be higher

when compared with those infected with E. coli

ESBL. Similarly, the different expression of

caspase-3 in the liver of rats, infected with those two

bacteria, may be caused by the soluble factors

secreted by both bacteria.

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