Detection of Antibiotic Residues in Chicken Meat and Eggs from Traditional Markets at Yogyakarta City Using Bioassay Method

Abstract

Studies on antibiotic residues content in food of animal origin are currently needed to support veterinary public health programs. The present study was described bioassay method for the detection of antibiotic residues in chicken meat and eggs from traditional market at Yogyakarta City. A number of twenty-four chicken meat samples and 24 egg samples were taken from 8 traditional markets in Yogyakarta city. Samples were examined at Centre for Veterinary Wates, Yogyakarta, Indonesia using bioassay method for screening detection of penicillin, aminoglycoside, macrolide and tetracycline residues. This bioassay method using some bacteria, such as Bacillus stearothermophillus, B. cereus, B. subtilis, and Kocuria rizophila. A percentage of the results showed that 8.33% (2/24) samples of chickens tested positively contained the oxytetracycline antibiotic residues. Meanwhile, as much as 75% (18/24) samples of positive eggs contain penicillin antibiotic residues, positive residues of aminoglycoside amounted to 12.5% (3/24) and the positive residues of oxytetracycline also amounted to 12.5% (3/24).

Full text

© 2019 Fakultas Kedokteran Hewan IPB http://www.journal.ipb.ac.id/indeks.php/actavetindones
ACTA VETERINARIA INDONESIANA
Special Issues: 1-6, December 2019
P-ISSN 2337-3202, E-ISSN 2337-4373
Research
Detection of Antibiotic Residues in Chicken Meat and Eggs from
Traditional Markets at Yogyakarta City Using Bioassay Method
Dyah Ayu Widiasih1*, Yatri Drastini1, Doddi Yudhabuntara1, F. Lintang R. Daru Maya2, Prisha Lini
Sivalingham2, Heru Susetya1, Widagdo Sri Nugroho1, M. Th. Khrisdiana Putri1, Roza Azizah Primatika1,
Bambang Sumiarto1
1Department of Veterinary Public Health, Faculty of Veterinary Medicine,
Gadjah Mada University, Indonesia
2Faculty of Veterinary Medicine,
Gadjah Mada University, Indonesia
*Corresponding author: dyahaw@ugm.ac.id
Submitted 6 November 2019, Accepted 22 December 2019
ABSTRACT
Studies on antibiotic residues content in food of animal origin are currently needed to support veterinary public
health programs. The present study was described bioassay method for the detection of antibiotic residues in chicken
meat and eggs from traditional market at Yogyakarta City. A number of twenty-four chicken meat samples and 24 egg
samples were taken from 8 traditional markets in Yogyakarta city. Samples were examined at Centre for Veterinary
Wates, Yogyakarta, Indonesia using bioassay method for screening detection of penicillin, aminoglycoside, macrolide
and tetracycline residues. This bioassay method using some bacteria, such as Bacillus stearothermophillus, B. cereus, B.
subtilis, and Kocuria rizophila. A percentage of the results showed that 8.33% (2/24) samples of chickens tested positively
contained the oxytetracycline antibiotic residues. Meanwhile, as much as 75% (18/24) samples of positive eggs contain
penicillin antibiotic residues, positive residues of aminoglycoside amounted to 12.5% (3/24) and the positive residues of
oxytetracycline also amounted to 12.5% (3/24).
Keywords: antibiotic residues, chicken meat, egg, bioassay
ABSTRAK
Studi tentang kandungan residu antibiotik dalam makanan yang berasal dari hewan saat ini diperlukan untuk
mendukung program kesehatan masyarakat veteriner. Penelitian ini menggambarkan metode bioassay untuk
mendeteksi residu antibiotik pada daging ayam dan telur dari pasar tradisional di Kota Yogyakarta. Sejumlah dua puluh
empat sampel daging ayam dan 24 sampel telur diambil dari 8 pasar tradisional di kota Yogyakarta. Sampel diperiksa di
Balai Besar Veteriner Wates, Yogyakarta, Indonesia menggunakan metode bioassay untuk mendeteksi residu penisilin,
aminoglikosida, makrolida, dan tetrasiklin. Metode ini menggunakan beberapa bakteri, seperti Bacillus
stearothermophillus, B. cereus, B. subtilis, dan Kocuria rizophila.
Persentase hasil menunjukkan bahwa sampel 8,3% (2/24) dari ayam yang diuji positif mengandung residu antibiotik
oxytetracycline. Sementara itu, sebanyak 75% (18/24) sampel telur positif mengandung residu antibiotik penisilin, residu
positif aminoglikosida sebesar 12,5% (3/24) dan residu positif oxitetrasiklin juga sebesar 12,5% (3/24).
Kata kunci: residu antibiotik, daging ayam, telur, bioassay

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INTRODUCTION
Chicken meat and eggs are food commodities of
animal origin-that are easily and commonly con-
sumed by the people of Indonesia. The increasingly
widespread use of antibiotics in poultry farms in-
creases the potential for antibiotic residues in chick-
en meat and eggs, and can trigger antibiotic re-
sistance or antimicrobial resistance (AMR). The
presence of antibiotics in foods of animal origin
needs to be monitored thoroughly since breeding,
because some antimicrobials have potential health
risks as shown in Table 1.
In past years, studies have been published that
antibiotic resistant bacteria were found in food of
animal origin (Ahlem et al., 2007, Levertein-van Hall,
et al., 2011, Geser et al., 2012, Ambrozic-Avgustin et
al., 2012). The impact of antibiotic residues on food
of animal origin can increase the potential threat to
toxicological, microbiological, and immunological
aspects in the human body. Among others, it can be
toxic to the liver, kidneys and central system of
haemopoietic, it also can disrupt the balance of mi-
croflora in the digestive tract and can trigger aller-
gies (Riviere and Papich, 2009). Food from animals
that contain antibiotic residues is still safe for con-
sumption as long as it is below the standard of max-
imum limit residue of permissible (MRL) (Table 2). In
Indonesia it has been regulated in SNI No. 01-
6366:2000 (BSN, 2000).
Meanwhile, the use of antibiotics as feed addi-
tives In Indonesia, are still widely common. Bahri et
al. (2006) reported that the use of tetracycline
and sulphonamide antibiotics as feed additives in
chicken by 74.43% (5 of 7) feed factories in Bo-
gor, Cianjur, Tangerang, Bekasi and Sukabumi
Regencies (Bahri et al., 2006). Meanwhile, Oramahi
et al. (2004) has also reported that chicken liver in
the city of Yogyakarta was contained antibiotics res-
idue respectively 29.23% for penicillin, 36.92% for
macrolide, 1.54% for macrolide and 26.15% for tetra-
cycline. The results of other research studies on the
assessment of the residues of several antibiotic
groups in chicken eggs in several provinces in Indo-
nesia showed that chicken eggs in Indonesia are still
relatively safe for consumption because no residual
content was detected in all egg samples tested
(Nurhidayah et al., 2015).
Therefore, since 2009 the Indonesian Govern-
ment as prohibited the use of antibiotics including
tetracycline as a feed additive through the Law
Number 18 of 2009 regarding Livestock and Animal
Health (Ministry of Agriculture Republic Indonesia,
2009).
Antibiotic residue testing can be done by rapid
testing (Wehr and Frank, 2004). Screening tests
with bioassays (Eennennaam et al.,1993, Pikkemaat
et al., 2009, BSN, 2008). ELISA test (Wang et al.,
2009) and by using High Performance Liquid Chro-
matography (HPLC) (Wehr and Frank, 2004)].
Screening tests with bioassays are qualitative tests,
which can be used easily, for large sample sizes, are
not too expensive and the results of false negative
tests are very small. ELISA and HPLC are common to
analyse antibiotic residue quantitatively and specific
to certain antibiotic (Zulfianti, 2005).
To ensure the food safety of food animal origin,
this study aims to reveal the level of antibiotic resi-
dues of chicken meat and eggs circulating in the
Yogyakarta region qualitatively by using bioassay
method.
Table 1 Main classes of antimicrobials and potential risks (J. European Comission, 2010)
Class
Health risks
Sulfamides
Allergies (with skin rashes), Sweet’s syndrome, DRESS syndrome, leukopenia
Quinolone
Immediate hypersensitivity reactions (urticaria, angioedema, anaphylaxis), exanthema,
Sweet’s syndrome
Beta-lactamines
Immediate reactions: urticaria, angioedema, rhinitis, bronchospasm and anaphylaxis, hae-
molytic anaemia, neutropaenia, eosinophilia.
Skin rashes, Stevens-Johnson syndrome, Lyell’s syndrome
Tetracyclines
Drug hypersensitivity syndrome, drug-induced lupus erythematosus such as a rash, anaphy-
laxis, DRESS syndrome, Sweet’s syndrome
Aminoglycoside
Allergic contact dermatitis
Phenicols
Rare bone marrow suppression: aplastic anaemia
Macrolides
Rare
Lincosamides
Neuromuscular blockade with post-anaesthetic paralysis, cardiac depression after too rapid
IV injection, allergies and moderate hepatic
degeneration

Detection of Antibiotic Residues | 3
© 2019 Fakultas Kedokteran Hewan IPB
MATERIAL AND METHODS
Sample Collection
Sampling was carried out from 8 traditional mar-
kets at Yogyakarta city by judgment method sam-
pling. The total chicken meat samples taken were
24, obtained from breast and thigh parts and total
eggs samples taken were 24. Sampling was carried
out aseptically. Furthermore, the samples were tak-
en to the Centre of Veterinary Wates, Yogyakarta,
with a coolbox for testing the presence of antibiotic
residues by bioassay method using Bacillus stea-
rothermophyllus, Bacillus cereus, Bacillus subtilis, and
Kocuria rizophila. During sampling, questioners were
also distributed to the sellers in the markets.
Screening Testing using Bioassay Method
Bioassay is carried out using microorganisms to
detect antibiotic compounds that are still active
(BSN, 2008). The principle of this test is that the
inhibition of bacterial growth by antibiotics
contained in food of animal origin shows a positive
effect on antibiotic residues (BSN, 2008). Bioassay
method on chicken meat and eggs to test the
content of antibioitic residues in this study was
carried out by weighing samples of chicken meat
and eggs each weighed as much as 10 g then added
buffer number 2 as much as 20 ml of phosphate
solution. Then homogenized using a homogenizer,
then centrifuged 3000 rpm for 10 minutes. The
supernatant is then taken and ready to be used as a
test sample solution (BSN, 2008).
Preparation
Antibiotic residues testing in chicken meat and
eggs using bioassay screening test methods refer to
SNI No. 7424: 2008, initiated with the making of
agar media, preparation of culture media, prepara-
tion of buffer solutions, and preparation of standard
solutions. The antibiotics used for this test were
penicillin, aminoglycoside, tetracycline and macro-
lide groups.
The media making for the antibiotic groups were
used peptone, yeast extract, bacto agar, aquadest,
and KH2PO4, each following the protocol in SNI.
Spesific bacteria were used for media culture prepa-
ration; for penicillin test used Bacillus stearother-
mophillus ATCC 7953, aminoglycoside test used B.
subtilis ATCC 6633, tetracycline test used B. cereus
ATCC 11778, and macrolide test used Kocuria rizophi-
la ATCC 9341 [13]. Buffer phosphate solution prepa-
ration also refer to SNI No. 7424: 2008 (BSN, 2008).
Samples Examination
At first, media cultured were prepared for each
antibiotic’s examination. Next, the petri dish was
added by 3 paper discs for each which one paper
disc has already soaked into sample extract, one
paper disc was dropped with standard solution as
positive control, and one paper dish was dropped
with buffer phosphate solution as negative control.
All paper discs were laid on precisely at the superfi-
cial of the agar media. The petri dishes then were
incubated in the incubator with the specific thermal
for each antibiotic. Tetracycline groups need incu-
bation thermal at 30°C ± 1°C, penicillin groups at 55°C
± 1°C, macrolide and aminoglycoside groups at 36°C
± 1°C along 16 to 18 hours. Each examination was
repeated three times (triple examination). The re-
sults of the assay method for testing antibiotic resi-
due were carried out by observing the inhibition
zones that formed after the incubation period was
completed and then measured in diameter using a
caliper. The inhibition zone is the working effect of
the presence of antibiotics which inhibits the
growth of bacteria around the paper disk. The di-
ameter of the inhibition zone formed shows the
concentration of antibiotic residues (Pikkemaat et
al., 2009).
Positive results if the inhibition zone formed is 14
mm ± 1 by using a 10 mm diameter paper disc or 12
mm ± 1 in diameter by using an 8 mm diameter pa-
per disc depending on the disc paper used.
Table 2 Maximum residue limit in chicken meat and eggs
Antibiotic
Maximum limit residue (mg/kg)
Penicillin
0.1
Oxytetracycline
0.05
Streptomycin
0.1
Erythromycin
0.1

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RESULTS
This study used 10 mm diameter paper discs, so it
would be positive if the inhibition zone formed were
≥ 14 mm ± 1. The results of the oxytetracycline resi-
due by using bioassay test from chicken meat sam-
ples are shown in Figure 1. The results of the antibi-
otic residue bioassay test from egg samples are
shown in Figure 2. The total results of tests on anti-
bacterial residues in chicken meat and eggs for sale
in traditional markets in the Yogyakarta region are
shown in Table 3.
There were 2 samples of chicken meat containing
oxytetracycline antibiotics and 3 samples of eggs
positive for antibiotics in the group of aminoglyco-
sides, 18 samples positive for penicillin, and 3 sam-
ples of eggs that were positive for antibiotic oxytet-
racycline.
DISCUSSION
The results obtained showed that the antibiotic
residues of penicillin, oxytetracycline and kanamycin
from the aminoglycoside group were found in
chicken and eggs sold in traditional markets in the
Yogyakarta region.
Qualitative test results using bioassay method on
chicken meat samples showed that 8.33% (2/24)
samples contained oxytetracycline. Meanwhile, the
test results of antibiotic residues in chicken eggs
obtained 75% (18/24) samples of chicken eggs con-
taining penicillin, oxytetracycline as much as 12.5%
(3/24) and contained aminoglycoside groups as
much as 12.5% (3/24) (Table 3). Meanwhile, the re-
sults of the questionnaire distributed to chicken and
egg traders could not be determined with certainty
because the traders only received from the company
Figure 1 Result of oxytetracycline residues by bioassay test in chicken meat samples. (a) Positive result as
inhibition zone formed were ≥ 14 mm ± 1. (b) Negative result as no inhibition zone were formed.
(c) Positive control with oxytetracycline. (d) Negative control versus positive control of macrolide
Figure 2 Result of Antibiotic residues by bioassay test in egg samples. (a) Positive result of pennicillin resi-
due. (b) Positive result of oxytetracycline residue. (c) Positive result of aminoglycoside residue
(kanamycin). (d) Negative control versus positive control of oxytetracycline

Detection of Antibiotic Residues | 5
© 2019 Fakultas Kedokteran Hewan IPB
or broiler chicken breeders and no one was raising
themselves.
The results above indicates that the use of anti-
biotics is still quite common in poultry farms in Yog-
yakarta area.
Oxytetracycline (OTC) is a broad-spectrum
antibiotic in the tetracycline class which is widely
used for the prevention and control of diseases in
poultry industry (Zulfianti, 2005). It can be used as
a respiratory treatment and if the dose is low it can
be used as a growth booster (Slana and Dolenc,
2013). Although the tetracycline group is only
allowed as animal medicine and is not included in
the feed additives that are permitted in Indonesia,
this class is often used as feed additives. It is
commonly used because of its many beneficial
aspects, including its availability, relatively
cheaper price, more easily use by oral
administration through drinking water or feed, can
increase the growth of broilers, and also can
increase the efficiency of feed use in broilers (Slana
and Dolenc, 2013, , Bachiri et al., 2017). The presence
of OTC in chicken meat is likely caused by it
characteristic, such as bacteriostatic; therefore it is
difficult to be metabolized and partly excreted in
the form of parent compounds due to its high
solubility in water (Bachiri et al., 2017).
The presence of high penicillin antibiotic residues
in eggs probably comes from the use of antibiotics
continuously and for a long time through drinking
water or feed in low concentrations. Penicillin can
interfere with the synthesis of bacterial cell walls,
consequently the bacterial cell wall ruptures. How-
ever, the use of penicillin that is too extensive is re-
sistance to anti-drug drugs. Some groups of bacteria
have antibiotic resistant properties, including Esche-
richia coli which can produce the enzyme penicilli-
nase so that it can damage penicillin. In addition,
some strains of Staphylococcus aureus and S. pneu-
moniae are also resistant to penicillin. Several strains
of Enterobacteriaceae are intrinsically aminopenicil-
lin-resistant, particularly among E. coli species
(Lobanovska and Giulia, 2017). Bachiri et al. (2017)
reported that African wildlife can act as a reservoir
of the epidemic E. coli clone ST131 producing CTX-M-
15 that indicated the presence of extended-
spectrum β-lactamase (ESBL)-producing Enterobac-
teriaceae in wild boars and Barbary macaques in Bé-
jaïa and Jijel, Algeria.
The results of this study are still qualitative and
need to be confirmed quantitatively by other meth-
ods such as HPLC, so that it can be known whether
the antibiotic residual content is still within the max-
imum standard residual limit or even exceeds so
that it can have an impact on human health. The re-
sults of this study also support previous studies that
chicken liver in the city of Yogyakarta was contained
mainly penicillin and tetracycline [9]. Nevertheless,
the results of our studies are of interest since food
animal origin, particularly chicken meat and eggs
were shown to be a potent reservoir of multidrug-
resistant organisms. Thus, the high prevalence of
antibiotic residues could be a source of resistance
developments among many bacterial strains. To de-
crease the resistance rate of bacteria, monitoring of
resistance, surveillance, prudent use, research pro-
jects, awareness, and educational programs are
recommended by WHO (2017). The application of
strict regulations on the use of antibiotics in the
field of chicken farming and monitoring the pres-
ence of residues on livestock products before they
are marketed is very necessary to prevent the ad-
verse effects of these residues on livestock origin
food products.
ACKNOWLEDGEMENTS
Researchers would like to thank Faculty of Veter-
inary Medicine, Gadjah Mada University for provid-
ing research funding. The researcher would also like
to thank the head of the Centre of Veterinary Wates
who had given the facility to conduct this study.
“All authors declare that there are no conflicts of
interest
⁠⁠⁠⁠
”.
Table 3 Antibiotic residues examination result
Number of positives from total sample (%)
Chicken meat samples
Egg samples
AMG*
MCR
PEN
OTC
AMG
MCR
PEN
OTC
-
-
-
2/24
(8.3)
3/24
(12.5)
-
18/24
(75)
3/24
(12.5)
Information: *AMG: aminoglycoside; MCR: macrolide; PEN: penicillin; OTC: oxytetracycline

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