Differences Expression of Caspase-3 In Hepar and Spleen of Rattus

norvegicus Infected with Methicillin Resistant Staphylococcus aureus

and Enterococcus faecalis

Sri Wahyuni

, Yoes Prijatna Dachlan.

and Agung Dwi Wahyu Widodo

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

Department of Anatomical Pathology, Faculty of Medicine, Universitas Airlangga,Surabaya, East Jawa, Indonesia

Department of Clinical Microbiology, Faculty of Medicine, Universitas Airlangga, Surabaya, East Jawa, Indonesia

Keywords: Caspase-3, Methicillin Resistant Staphylococcus aureus (MRSA), Entrerococcus faecalis

Abstract: Bacteria are organisms that can cause infection. Methicillin Resistant Staphylococcus aureus (MRSA) and

Entrerococcus faecalis are gram-positive bacteria that can cause nosocomial infections. Virulence factors of

MRSA thus play a role in the infection process, such as are polysaccharide, surface proteins such as

adhesins, glycoprotein, hemagglutinin, and fibronectin, while enterococcus such as gelatinase, cytolysin,

enterococcal surface protein (Esp) and aggregation substance (AS) are the virulence factors of Enterococcus

that can cause infection. This research is True Experimental research with post-test only for the Control

Group Design. The MRSA and E. faecalis bacteria were injected intraperitoneally to R. norvegicus and

observed after 24 hours. Animals were placed into three groups: the control group, treatment with MRSA,

and Enterococcus faecalis. The hepar and spleen were isolated from the dead R. norvegicus and a

Immunohistochemistry (IHC) test was conducted to observe the expression of caspase-3 by light

microscope. The result showed that the caspase-3 expression increased in the infected group of MRSA and

Enterococcus faecalis compared with the control group. The expression of caspase-3 in the hepar was

higher than in the spleen. The hepar serves as the receiver of the portal and systemic circulation. The hepar

also plays an important role in the host defense that is exposed to and will catch pathogens, followed by

cleaning. The increased expression of caspase-3 suggests the cell death also increased.

1 INTRODUCTION

Infectious diseases still occupy the top causes of

morbidity and mortality in developing countries

(Triana, 2014). Bacteria is one of the organisms that

can cause infectious diseases (Kaufmann et al.,

2011). The spread of diseases is caused by various

intermediaries, including air, animals, objects,

humans themselves, and even unconsciously, the

hospital becomes a high-risk source of transmission

(Triana, 2014). These bacteria include MRSA

(Sandi, et al., 2015) and Enterococcus faecalis

(Chen and Zervos, 2009).

Increased incidence of the S. aureus infection,

especially MRSA with the phenomenon of antibiotic

resistance, is considered as one of the biggest

barriers to infection control. MRSA is a bacteria that

causes nosocomial infection (Sandi et al., 2015) and

Enterococcus faecalis (Tortora, 2016). Enterococcus

faecalis is a commonly found species of

Enterococcus (Chen and Zervos, 2009). MRSA and

Enterococcus faecalis are gram-positive bacteria that

can cause infections involving the death of cells

(Chen and Zervos, 2009; Sandi et al., 2015).

Apoptosis is the programming of cell death used

to prevent inflammation and limit cell damage

(Martinez, 2017). Increased toxins, produced by

bacteria, will increase apoptosis (Baudouin, 2008).

Apoptosis is also used for homeostasis and

protection against bacterial infections (Upton and

Chan, 2014).

The hepar is an organ that plays an important

role in the defense of the host against the invasion of

microorganisms (Talwani et al., 2013), while the

spleen is the main filter for pathogens and antigens

carried by blood. The spleen is an area of regulation

host immune response to initiate innate and adaptive

immune responses to pathogens and the

establishment of specific antigens in immune

Wahyuni, S., Dachlan, Y. and Wahyu Widodo, A.

Differences Expression of Caspase-3 In Hepar and Spleen of Rattus norvegicus Infected with Methicillin Resistant Staphylococcus aureus and Enterococcus faecalis.

DOI: 10.5220/0007540902530257

In Proceedings of the 2nd International Conference Postgraduate School (ICPS 2018), pages 253-257

ISBN: 978-989-758-348-3

253

responses that protect bacterial, viral, and blood-

borne fungal infections (Bronte and Pijtet, 2013).

Caspase is the leading cause of cell death by

dividing cell proteins to disassemble cells that will

die. In the arrangement, it is important that caspase

be maintained to avoid unexpected cell death

(Parrish et al., 2013). Caspase-3 is a member of the

caspase family that plays a role in the execution

phase in the apoptosis (Prakosa et al., 2013).

Caspase-3 is also the best effector caspase and an

apoptotic signal, if there is activation of caspase-3,

there is no cell rescue (Parrish et al., 2013).

2 MATERIALS AND METHOD

2.1 Animal model

The animal model used in the research was the

Rattus novergicus with the criteria of being male,

healthy, age three months, and a body weight of

150–200 grams.

2.2 Bacteria

The bacteria of MRSA and Entrerococcus faecalis

were obtained from the Installation of Clinical

Microbiology RSUD Dr. Soetomo Surabaya. The

concentration was 10

CFU bacteria with Phosphate-

buffer saline (PBS).

2.3 Sample

The animal models were placed in three groups,

each consisting of four rats. The groups were

infected with MRSA and Entrerococcus faecalis, as

a control were given PZ (1 ml in the peritoneum of

each rat; the concentration was 10

CFU bacteria

with PBS). Observation was performed for 24 hours

post infection. Rats were sacrificed for removal of

the hepars and spleens. Organs were fixed in

formalin buffer and preparation (formalin fixed and

paraffin embedded section were performed) and

Immunohistochemistry for Caspase-3 using a rabbit

and mouse antibody. The expression cells were

observed under a light microscope with an objective

100x in a five field of view. The analysis of data

using was carried out using GraPhad prism.

3 RESULTS

3.1 Expression of Caspace-3 in Hepar

Of the mean number of the cells expressed in the

caspase-3 in hepar, the group infected with MRSA

were higher than the group infected with

Entrerococcus faecalis (Figure 1).

Figure 1 : Bar-graph Expression of Caspase-3 in hepar.

The highest expression of caspase-3 that infected

was the MRSA group (96.75%), than the

Entrerococcus faecalis (84.25%), and finally the

control group (9.5%) (Figure 1).

Figure 2 : Expression of caspase-3 in hepar, (a) control

group of R. norvegicus, (b) Entrerococcus faecalis

group,

ICPS 2018 - 2nd International Conference Postgraduate School

254

3.2 Expression of caspase-3 in the Spleen

The mean number of cells was expressed by

caspase-3 in the spleen; the group infected with

MRSA were higher than the group infected with

Entrerococcus faecalis (Figure 3).

ntrol

MRS

E. f

ecalis

Expression of Caspase-3 (%)

Figure 3 : Bar-graph Expression of Caspase-3 in the

Spleen.

The highest expression of caspase-3 was MRSA

(19.25%) then Enterococcus faecalis (11.5%), and

then the control group (4.25%) (Figure 4).

Figure 4 : Expression of caspase-3 in the spleen, (a)

control group of R. norvegicus, (b) Entrerococcus faecalis

group, (c) MRSA group. Magnification x1000.

4 DISCUSSION

Apoptosis plays a major role in removing infected

cells, mutating (or causing damage during

development), tissue homeostasis, and the effects of

aging. Apoptosis is associated with morphological

and biochemical changes, including the release of

cytochrome c, the activation of protease caspase,

chromatin condensation, and DNA fragmentation

(Jafari et al., 2015).

The virulence of MRSA plays a role in an

infection, such as polysaccharides and surface

proteins (Gordon and Lowy, 2008). The surface

protein is responsible for colonization.

Polysaccharides and protein-A are inhibit

phagocytosis by polymorphonuclear leukocytes. The

enzyme catalase is also a factor that supports the

bacteria for survival inside phagocytic cells (Sandi et

al., 2015). Bacteria produces various virulence

factors that allows them to escape from the body’s

immunity, infecting and spreading to remote organs

(Ramachandran, 2014).

The Virulence of Enterococcus faecalis causes

infections such as gelatinase, enterococcal surface

protein (Esp), aggregation substance (AS) and

cytolysine (Uysal, 2013). Cytolysine can lyse

various target cells; it can lyse erythrocyte cells,

polymorponuclear neutrophils, and macrophages.

Cytolysine may also increase toxicity and

bacteriocin activity for colonization. Other factors,

such as gelatinase may help bacteria to avoid the

immune system (Tyne et al., 2013). Aggregation

substance promotes direct binding, independent

opsonin to polymorphonuklear leukococyte can

survive in different phagocytes. Enterococcal

surface proteins with multiple repetitive motives in

the encoding gene may be important in immune

avoidance. Products that cause direct tissue damage

are cytolysine and gelatinase (Waar, 2004).

Apoptosis can within or outside the host cell

because of stimulation of microbial infections,

oxidative stress, DNA damage or DNA errors or

when the cells have reached the final cycle. Started

enzymes form a cascade signal, send a danger signal

through the caspase initiator and continue to capase-

3 as the executor. The enzyme initiates cell

disassembly by degrading the DNA. The toxins of

bacteria cause inflammation and increased caspase-3

and apoptosis (Wall and Beth, 2014). The molecular

strategies by bacteria to interact with the host can be

unique to a particular pathogen (Wilson et al., 2002).

Hepar is the portal defense for bloodstream

infections. Hepar contains immune cells to bind and

clean pathogens (Talwani et al., 2013). The spleen is

Differences Expression of Caspase-3 In Hepar and Spleen of Rattus norvegicus Infected with Methicillin Resistant Staphylococcus aureus

and Enterococcus faecalis

255

the filter for pathogens and the antigen carried in

blood. Establishment of the immune system to

specific antigens from bacterial, viral, and fungal

infections. The spleen uses the regulation of immune

response (Bronte and Pijttet, 2013) and filters to

remove blood cells due to aging or pathological

changes (Pivkin et al., 2016).

MRSA and Enterococcus are bacteria that can

cause tissue damage especially for hepar. During

infection in the bloodstream, the majority of bacteria

are sequestered immediately by Kupffer cells in the

hepar and increased enzymes in the hepar

(Kolaczkowska et al., 2015). Abnormal enzyme

levels may indicate hepar damage (Giannini et al.,

2005). Response hepar damage to infection is occurs

increased caspase-3 and apoptosis (Kolaczkowska et

al., 2015).

5 CONCLUSIONS

In conclusion, there was an increase of expression

caspase-3 in the hepar and spleens of R. norvegicus

when the group infected with MRSA was higher

than that infected with Enterococcus faecalis.

Increased expression of caspase-3 suggests the cell

death also increased.

ETHICS APPROVAL

All documents for ethics approval as well as

proposal of the research have been reviewed by the

ethics committee of Universitas Airlangga Faculty

of Dental Medicine, as described on the ethical

approval No. 265/HRECC.FODM/X/2017.

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and Enterococcus faecalis

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