The Detection of Staphylococcus aureus in birdnest

The Detection of Staphylococcus aureus in Swiftlets' Nest
Folia Medica Indonesiana Vol. 266 41 No. 4 October – December 2005
THE DETECTION OF Staphylococcus aureus IN SWIFTLETS' NEST
USING IMMUNOHISTOCHEMISTRY (STREPTAVIDIN BIOTIN)
Retno Oktorina*, Soedarmanto Indarjulianto**, Sitarina Widyarini**, Hastari Wuryastuti**, R. Wasito**
ABSTRACT
A study to detect the presence of Staphylococcus aureus in swiftlets' nest using immunohistochemistry (Streptavidin
biotin Complex) has been successfully done. Tissue and supernatant were made from the nest, and the presence of the
bacteria Staphylococcus aureus was detected by means of immunohistochemical method. As positive control, we used
Staphylococcus aureus culture, while for negative control we replaced Staphylococcus aureus monoclonal antibody
with Phospat Buffer Saline (PBS). The result showed that staining with Staphylococcus aureus monoclonal antibody in
swiftlets' nest tissue revealed the presence of Staphylococcus aureus as a brownish group or cluster, resulting from the
reaction of enzymes and chromogen in Streptavidin Biotin Complex. Based on this study, it can be concluded that
immunohistochemical method (Streptavidin Biotin Complex) can be used to detect the presence of Staphylococcus
aureus in swiftlets' nest.
Keywords: swiftlets' nest, S. aureus, immunohistochemistry
INTRODUCTION
A major challenge for Indonesia is to produce animal
food products which are safe for consumers' health.
Animal food safety is not only the world's issue
(Anonym, 2000), but also every indivual's concern. It is
a consumer's right to have safe animal food. Indonesia is
the largest producer and supplier of swiftlets' nest, with
Hongkong, USA, Singapore, Malaysia, China, Japan,
and UK as the major export destinations (Iswonto,
2002). Playing the role as largest producer, Indonesia
should maintain the aspect of food quality as the main
consideration in trade. Market requirement and product
suitability for consumers should be met by increasing
product acceptability and competitiveness in global
market (Anonym, 2000).
Swiftlets' nest is an exotic or delicate food. In addition
as a delicious serving, it can also be used as material for
medications that improves physical strength (Winarno,
1994; Budiman, 2002; Iswanto, 2002). As in other food
materials, swiftlets' nest may subject to damage
resulting from pesticide residuals, animal drugs, heavy
metals, other contaminants, as well as the growth of
microbes, such as bacteria, virus, yeast, and fungi,
which may cause food-borne disease.
To support the availability of safe food products as the
basic consideration in trade, we need microbial
detection method for swiftlets' nest. In a preliminary
______________
*Animal Quarantine Center, Tanjung Perak, Surabaya
**Gadjah Mada University School of Veterinary
Medicine, Yogyakarta
study, Animal Quarantine Board (Balai Karantina
Hewan) in cooperation with Airlangga University
School of Pharmacy had undergone a test on
Microbiological Quality Control for export swiftlets'
nest in Animal Quarantine Juanda (unpublished data).
The result of this preliminary study, obtained using
rapid test (Oxoid, United Kingdom) and followed with
fertilization in agar media, showed that Staphylococcus
spp was identified in five samples of swiftlets' nest,
while Escherichia coli was identified in one sample.
Salmonella spp and Pseudomonas spp were not found.
Staphylococcus spp is a group of bacteria that plays an
important role in food microbiology, and
Staphylococcus aureus is the prominent bacteria in food
because during its growth the organism can produce
enterotoxin. Ecologically, Staphylococcus aureus is
closely related with human beings. In largest amount of
cooked or salted foods, Staphylococcus aureus can
unceasingly grow until reaching a hazardous level
(Buckle, 1987). Based on this preliminary study, a fast
and accurate method to detect Staphylococcus aureus in
swiftlets' nest was needed. The method that is recently
developing is the use of immunohistochemistry by
means of the principle of specific binding between
antigen and antibody which was visualized through
enzymes and substrates. This method used basic
principles of immunology in tissue or cells.
The Detection of Staphylococcus aureus in Swiftlets' Nest
Folia Medica Indonesiana Vol. 267 41 No. 4 October – December 2005
MATERIALS AND METHODS
This study used swiftlets' nest samples ready to be
exported through Juanda Airport. The samples were
processed to make preparations in Veterinary Disease
Inspection Bureau (Balai Penyidikan Penyakit
Veteriner, BPPV) Regional IV Yogyakarta in
accordance with standard procedure of BPPV
laboratory. The swiftlets' nest preparation in paraffin
embedded tissue section was put onto Poly-L-lysin
(SIGMA)-coated glass object. The
immunohistochemical staining used Streptavidin Biotin,
with stages as recommended by Wasito (1997). The
swiftlets' nest preparation was paraffinized by giving (a)
xylene, three times each for 2 minutes, (b) 100%
ethanol, twice each for 2 minutes, (c) 95% ethanol, once
each for 2 minutes, (d) 50% ethanol, each for 2 minutes,
and (e) distilled water, twice each for 2 minutes, and
Phosphate Buffer Saline (PBS) of 0.01 m with pH 7.1
for 5 - 10 minutes. Subsequently, the preparation was
immersed in H2O2 to remove endogeneous peroxidase,
and incubated in a microwave. The preparation was then
washed with PBS for 10 minutes, and incubated with
blocking serum (V Block) solution for 10 minutes. The
excessive serum was removed from the preparation and
the latter was directly given with primer antibody, i.e.
Staphylococcus aureus monoclonal antibody and
incubated at room temperature for 45 minutes, and
washed with PBS for 10 minutes. Antigen retrieval was
done using citric acid and microwaved for 10 minutes
(Shan et al, 1997). The preparation was incubated with
Biotynilated Secondary Antibody (Lab Vision, USA) at
room temperature for 10 minutes, incubated with
chromogen substrate (Lab Vision, USA) at room
temperature for 15 minutes, washed with distilled water,
and mounted with glycerol to be observed under the
microscope.
For culture preparation of the isolates of Staphylococcus
aureus and supernatant from swiftlets' nest sample, the
stages of immunohistochemical staining were the same
as those in paraffin-embedded tissue section. The
difference was that in supernatant preparation, the
swiftlets' nest should be paraffinized and washed
directly for 5 minutes, while the rest of the procedures
were all the same. To obtain supernatant preparation, 5
grams of swiftlets' nest sample were finely ground,
added with physiologic NaCl and left overnight.
Subsequently, the preparation was dripped on Poli-Llysine-
coated glass object, and incubated in microwave
for 12 hours and subjected to immunohistochemical
staining using Streptavidin Biotin method (Wasito,
1997). As positive control for this staining method, we
used Staphylococcus aureus colony. Negative control
was made by replacing primary antibody with
Phosphate Buffer Saline (PBS).
RESULTS AND DISCUSSION
The objective of this study was to apply
immunohistochemical method by using Streptavidin
Biotin Complex. This technique is a modification of
indirect method, in which one antigen from swiftlets'
nest is bound by antibody in two stages. First, the
primary antibody is directly bound to antigen.
Afterwards, the antibody will be bound to biotinilyzedprimary
antibody. The binding between antigen and
antibody would be visualized by the change of enzymes
and substrates into brownish color (Hoffman, 1996;
Wasito, 1997; Harkow F and Lane, 1999). The
application of immunohistochemical method is
immunohistochemical staining to culture, supernatant,
and swiftlets' net tissue. Immunohistochemically-stained
Staphylococcus aureus culture from the nest revealed
brown precipitation, indicating the binding between
antigen and antibody as visualized through the reaction
of peroxidase and 3,3 diaminobenzidine
tetrahydrochloride (Figure 1). The Staphylococcus
aureus looks grouped or clustered.
Immunohistochemically-stained swiftlets' nest
supernatant showed the presence of antigen
(Staphylococcus aureus) and antibody binding, which
was visualized by the presence of brown precipitation
(Figure 2). This was in line with the basic principle of
chromogen, a marker that can visualize marker
substance at immunocomplex binding in
immunohistochemical staining. In this principle, the
binding between chromogen and peroxide (marker
substance) is visualized brown by using 3,3
diamonobenzidine tetrahydrochloride chromogen
(Baroff and Cook, 1994; Wasito, 1997). The paraffin
embedded tissue section of swiftlets' nest using
Streptavidin Biotin method (Lab Vision, USA) showed
the result of antigen (Staphylococcus aureus) and
antibody binding visualized as having brown color
(Figure 3).
The Detection of Staphylococcus aureus in Swiftlets' Nest
Folia Medica Indonesiana Vol. 268 41 No. 4 October – December 2005
Figure 1. Staphylococcus aureus culture in swiflet’s nest
Figure 2. Staphylococcus aureus supernatant in swiflet’s nest
Figure 3. Swiflet’s nest tissue
The Detection of Staphylococcus aureus in Swiftlets' Nest
Folia Medica Indonesiana Vol. 269 41 No. 4 October – December 2005
Those results showed that immunohistochemical
method using Streptavidin Biotin can be used to detect
Staphylococcus aureus in supernatant and swiftlets' nest
tissue. Previous studies were reported by Cleary et al
(2004), Priambodo (2004) and Tsusumi et al (1991)
who detected bacteria in intestinal epithelium, blood and
urine using immunohistochemical staining. In these
studies the bacteria was apparent in the form of cluster
or clump (bacterial coated antibody).
In culture preparation using immunohistochemical
staining (Streptavidin biotin), the supernatant and
preparation from swiftlets' nest tissue had a brown
color, a result of binding between antigen
(Staphylococcus aureus) and its monoclonal antibody
which was visualized through chromogen substrate in
the colony of the bacteria that formed a group or cluster
(Duguid, 1989).
CONCLUSION
Immunohistochemical staining can be used to detect the
presence of Staphylococcus aureus in swiftlet's nest.
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