- Email: [email protected]
Screening of quinolone antibiotic residues in chicken meat and beef sold in the markets of Ankara, Turkey
Screening of quinolone antibiotic residues in chicken meat and beef sold in the markets of Ankara, Turkey Buket Er,*1 Fatma Kaynak Onurdağ,† Burak Demirhan,* Selda Özgen Özgacar,† Aysel Bayhan Öktem,* and Ufuk Abbasoğlu† *Department of Food Analysis, and †Department of Pharmaceutical Microbiology, Faculty of Pharmacy, Gazi University, 06330 Ankara, Turkey ABSTRACT This study aimed to find the effects of quinolone antibiotics in chicken and beef used in Ankara, Turkey. Total number of 127 chicken and 104 beef meat samples were collected randomly from local markets for analysis. Extraction and determination of quinolones were made by ELISA procedure. One hundred eighteen of 231 (51.1%) examined chicken meat and beef samples were found to contain quinolone antibiotic residue.
Among the chicken meat and beef samples, 58 (45.7%) of chicken meat samples and 60 (57.7%) of beef meat samples were positive for quinolones, respectively. The mean levels (±SE) of quinolones were found to be 30.81 ± 0.45 μg/kg and 6.64 ± 1.11 μg/kg in chicken and beef samples, respectively. This study indicated that some chicken and beef meat sold in Ankara contains residues of quinolone antibiotics.
Key words: antibiotic, chicken, enzyme-linked immunosorbent assay, quinolone, residue 2013 Poultry Science 92:2212–2215 http://dx.doi.org/10.3382/ps.2013-03072
INTRODUCTION Many antimicrobial agents are used in farm animals for therapeutic and prophylactic purposes, and as a result of this application, trace quantities of the drugs or their metabolites may be present as residues in food. The residues of these substances or their metabolites in meat and other foods of animal origin may cause adverse effects on consumers’ health (Moreno-Bondi et al., 2009). The common use of antimicrobial agents for therapeutic and nontherapeutic purposes has been mentioned as a reason for the occurrence of antibiotic resistance among human beings (Ghosh and LaPara, 2007), and thus, subtherapeutic use of these antimicrobial agents in animals is considered to have an impact for gaining resistance among environmental bacteria (Bester and Essack, 2010). Therefore, the analysis of the residues of these compounds in meat and meat products is very important for human health (Mahgoub et al., 2006). In recent years, a public health consequence of the use of antibiotics in food animals has grown (Jafari et al., 2007). Foodborne bacterial pathogens are resistant to antibiotics, resulting in increased human morbidity and mortality (Wegener, 1999).
©2013 Poultry Science Association Inc. Received January 28, 2013. Accepted May 10, 2013. 1 Corresponding author: [email protected]
Quinolones and fluoroquinolones are important antibiotics used in human and veterinary medicine (Velissariou, 2006; Andreu et al., 2007; Cañada-Cañada et al., 2009; Cháfer-Pericás et al., 2010). Quinolones may have a directly toxic effect or lead to the emergence of drug-resistant bacteria predicating a potential risk to human health. These residues can produce allergic hypersensitivity reactions or toxic effects (Juan-García et al., 2006; Cañada-Cañada et al., 2009). Presently, several quinolones are acceptable for treatment of beef, poultry, and fish in many countries of the world (WHO, 1998). In poultry, antibiotics are used for reducing the incidence of disease and for growth promotion. Antibiotics promote growth by decreasing activity of the immune system and reducing waste of nutrients and toxin formation. Unfortunately, contaminated poultry tissues with harmful concentrations of drug residues may occur (Donoghue, 2003; Nisha, 2008; Shareef et al., 2009). Quinolone is one of the most widely used antibiotics in veterinary medicine for treatment and prevention of diseases. Residues of quinolone may be observed in foods of animal origin if proper checks and balances are not employed. It has been reported that, because of the presence of quinolone residues in human food obtained from animal sources, effectiveness of quinolones in human treatment has decreased (Donkor et al., 2011). In Turkey, ciprofloxacin, enrofloxacin, marbofloxacin, danofloxacin, difloxacin, sarafloxacin, flumequin, and oxolinic acid are approved quinolones for usage in ani-
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SCREENING OF QUINOLONE ANTIBIOTIC RESIDUES
mal production. Among these quinolones, sarafloxacin is permitted in skin, wing meat, and liver of chicken but not in breast meat. According to the Turkish Food Codex (TFC), the maximum residue levels of the sum of enrofloxacin and ciprofloxacin in chicken muscle and beef muscle should not exceed 100 µg/kg; marbofloxacin should not exceed 150 µg/kg in beef muscle; danofloxacin should not exceed 200 µg/kg in chicken muscle and beef muscle; difloxacin should not exceed 300 µg/ kg in chicken muscle and 400 µg/kg in beef muscle; flumequin should not exceed 400 µg/kg in chicken muscle and 200 µg/kg in beef muscle; and oxolinic acid should not exceed 100 µg/kg in food animal muscle (TFC, 2002, 2011). The aim of this study was to determine the quinolone residues in chicken meat and beef samples collected from Ankara region local markets.
MATERIALS AND METHODS Sample Collection In this study, 127 chicken breast meat and 104 beef round meat samples were collected from local markets in 3 different districts (A, B, C) of Ankara, Turkey. Between July 2010 and September 2010, chicken meats were collected, and between September 2010 and October 2010, beef meats were collected. Among beef round samples, 50 samples originated from domestic animals and 54 samples originated from foreign animals. The samples were preserved at 4°C soon after collection. Methanol was purchased from Merck (Darmstadt, Germany).
Analysis of Samples Quinolone residues were determined with an ELISA using the Ridascreen Quinolones/ELISA kit (RBiopharm AG, Darmstadt, Germany). The assay was performed according to the guidelines of the manufacturers. The ELISA kit showed cross-reactivity with ciprofloxacin (100%), norfloxacin, enrofloxacin, marbofloxacin, danofloxacin, difloxacin, flumequin, ofloxacin (>100%), sarafloxacin (43%), and oxolinic acid (24%). The detection limit was reported as 5 µg/kg, being the lowest value for chicken meat and beef in the test kit. Sample preparations were done according to the instructions of the Ridascreen kit. Briefly, each of randomly selected muscle tissue sample was homogenized using a Turrax disperser and 5 g of the homogenized sample was transferred to centrifuge tubes, containing 4 mL of methanol/water (70:30, vol/vol) and vortexed for 10 min. Subsequently, this was centrifuged at 4,000 × g for 10 min. Fifty microliters of the standard solution and each of the samples were added to 96-well microplates. Fifty microliters of enzyme conjugate and 50 µL of antibody were added to the wells, respectively, and mixed gently and incubated for 1 h at 4°C. At the end of the incubation the liquid was poured from the wells and washed twice with the washing buffer. One
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hundred microliters of the substrate/chromogen was added to the wells and incubated at room temperature for 15 min. One hundred microliters of the stop solution was added to each well and the absorbance was measured at 450 nm in ELISA plate reader (VersaMax Tunable). Quinolones concentrations were calculated through the guidelines of the Ridascreen kit (R-Biopharm AG). Calibration curves were plotted as semilogarithmic concentration versus the ratio of the mean absorbance at each concentration divided by the mean absorbance of the zero standard (B/Bo). The B/Bo values were decreasing, which is a good result for the reliability of the test. The results of the samples were calculated after the B/Bo values were evaluated.
Statistical Analyses Independent sample t-test and Shapiro-Wilk test were conducted for statistical comparison.
RESULTS AND DISCUSSION One hundred eighteen of 231 (51.1%) examined chicken meat and beef samples were found to contain quinolone antibiotic residue. Fifty-eight (45.7%) of the chicken meat samples were positive for quinolones, and 60 (57.7%) of beef meat samples were positive for quinolones. The mean levels of quinolone antibiotic residue were found to be 30.81 ± 0.45 µg/kg in positive chicken samples. However, quinolone residues were found in low levels in beef samples. The mean levels of quinolone residues were as low as 6.64 ± 1.11 µg/kg in positive beef samples. Our data revealed that quinolone mean levels in chicken and beef meat samples were lower than 100 µg/kg, which is the lowest residue value mentioned in the TFC. This study indicated that some chicken and beef meat sold in Ankara contains residues of quinolone antibiotic, although these levels were low. In 2 of chicken samples, a level of quinolone was determined to be higher than 100 µg/kg. These levels were determined as 103 and 106.2 µg/kg. The concerning values are shown in Tables 1 and 2. Limited studies on antibiotic residues in poultry animals have been performed in Turkey. However, considering the importance of antibiotic residues in foodstuffs, periodic sampling is carried out in many countries (Salehzadeh et al., 2007; Weiss et al., 2007; Zhao et al., 2009; Pena et al., 2010). Akar (1994) reported 175 chicken meat samples in Ankara and found antibiotic (chloramphenicol, erythromycin, and tylosin) residue rate to be 5.7%. Yüksek (2001) reported 50 chicken meat samples in Ankara and did not find residue of oxytetracycline, chloramphenicol, and zinc bacitracin in muscle tissues of the chicken. Pena et al. (2010) analyzed 98 samples of chicken samples in the north and central zones of Portugal and found fluoroquinolones residue rates to be 44.2%. Zhao
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Er et al. Table 1. Statistical analyses for levels of quinolone in positive samples of beef meat1 Origin of beef meat
Sample tested, n
Positive samples, n (%)
Concentration of positive samples, µg/kg (mean ± SE)
Foreign Domestic Total
54 50 104
33 (61.1) 27 (54.0) 60 (57.7)
6.74 ± 0.21 6.50 ± 0.20 6.64 ± 0.14
1There is no statistically significant (P > 0.05) difference between foreign and domestic beef meats in case of quinolone concentrations.
et al. (2009) determined 11.7% quinolone residue rate in 14 of 120 chicken muscle samples and found quinolone residue levels of 0.7 to 43.6 ng/g. Salehzadeh et al. (2007) determined 270 chicken muscle, liver, and kidney samples from 90 broiler farms in Tehran, Iran, and found enrofloxacin mean concentrations as 18.32 ± 32.29, 18.34 ± 12.36, and 26.06 ± 19.52 ng/g in muscle, liver, and kidney samples, respectively. Weiss et al. (2007) analyzed 299 samples of poultry meat in Italy and could not found enrofloxacin antibiotic residue. These contamination rates in chicken samples were lower in comparison with values reported in this study. The literature available on the occurrence of antibiotics in poultry samples indicates higher rates of antibiotic residues. Shareef et al. (2009) reported antibiotic residue rate as 52% in poultry products from a total of 75 samples. Naeem et al. (2006) analyzed poultry products in Pakistan and reported that 58 to 85% of the samples contained ciprofloxacin residues and 55 to 92% of the samples contained enrofloxacin residues in violation of the regulations. In Turkey, there is limited number of studies related to the search for antibiotics in cattle. Kaya et al. (1992) reported the antibiotic residue rate as 18% in samples from a total of 85 cattle in Turkey. Koç (2006) analyzed 240 samples of cattle and sheep meats in Ankara, Turkey, and did not find quinolone antibiotic residue. These results in cattle samples show compatibility with the results of this study. Quinolone antibiotic residues contamination is also reported in milk samples collected from Turkey. Kaya and Filazi (2010) determined 1.25% antibiotic residue contamination rates in milk samples. Alla et al. (2011) analyzed beef samples in Sudan and reported that 3% of the muscles contain antibiotic residue. Donkor et al. (2011) reported antibiotic residue rate of 30.8% in samples from a total of 156
beef samples in Ghana. Masztis (1984) analyzed 487 bovine carcasses in Canada and found that 12 samples were positive. Antibiotic residues can be found at different concentration levels in foods of animal origin such as fish, beef, poultry, pork, milk, and milk products (De Wasch et al., 1998; Vragović et al., 2011). Consequently, antibiotic residues have been indicated as a public health concern. Quinolones are potent inhibitors of DNA gyrase enzyme, which is critical for DNA replication and transcription (Suto et al., 1992). The toxicity and side effects of quinolones are well established in animals and humans (Christian, 1996; Mäkinen et al., 1997; Shimoda, 1998; Khadra et al., 2012). Fluoroquinolones show damage to the juvenile joint, the kidney, the eye, and the central nervous system, which has been previously reported by animal experiments (Patterson, 1991). Some antibiotic-induced allergic reactions have also been reported, in relation to quinolones (Gruchalla and Pirmohamed, 2006). Given the adverse effects of quinolones on human health, residues in food and other environmental sources are also expected to affect human health. Therefore, it is necessary to monitor frequently consumed meat and meat products with high nutritional value for the presence of residual quinolones. The analysis of residues of these drugs in edible tissues is very important for human health. In Turkey, using quinolone for the prophylaxis and treatment of chicken and beef is legal, but there is a limit in the preslaughter withdrawal times. Also, there is a limit for the quinolone residues in chicken and beef meat in the TFC. According to these limitations, low levels of quinolone residues in chicken and beef meat is not a surprising but a good result. These results are valid for the obtained samples during the study. Results may change periodically. Therefore,
Table 2. Statistical analyses for levels of quinolone in positive samples of chicken meat
District A B C Total
Samples tested, n 41 56 30 127
Positive samples, n (%) 12 37 9 58
(29.3) (66.1) (30.0) (45.7)
Concentration of positive samples, µg/kg (mean ± SE) 15.23 36.65 27.58 30.81
± ± ± ±
2.22 4.04 7.30 3.04
Mean value of samples 14.03a 36.03b 16.33a
a,bFor an attribute, means within a groups in a column (between different districts) not having a common superscript letter are different (P < 0.05).
SCREENING OF QUINOLONE ANTIBIOTIC RESIDUES
the steps in food processing should be kept under continuous monitoring for preventing overexpressed levels of quinolone residues. In conclusion, the chicken and beef samples obtained during the period of the study are thought to exclude health risks in terms of quinolone presence. In terms of preventing antibiotic resistance in humans, the low amount of quinolone residue levels observed in this study represents a positive result for local food control.
ACKNOWLEDGMENTS This study was partly supported by Gazi University Research Foundation (project no. 02/2010-24). The authors are thankful to Khalid Mahmood Khawar, Department of Field Crops, Faculty of Agriculture, Ankara University, for guidance and preparation of the manuscript.
REFERENCES Akar, F. 1994. Investigation on various antibiotic residues in chicken meat and liver. Vet. J. Ankara Univ. 41:199–207. Alla, M. B. W., T. E. Mohamed, and A. E. Abdelgadir. 2011. Detection of antibiotics residues in beef in Ghanawa Slaughterhouse, Khartoum State, Sudan. AJFS 5:574–580. Andreu, V., C. Blasco, and Y. Pico. 2007. Analytical strategies to determine quinolone residues in food and the environment. Trends Analyt. Chem. 26:534–556. Bester, L. A., and S. Y. Essack. 2010. Antibiotic resistance via the food chain: Fact or fiction? S. Afr. J. Sci. 106:1–5. Cañada-Cañada, F., A. Muñoz De La Peña, and A. Espinosa-Mansilla. 2009. Analysis of antibiotics in fish samples. Anal. Bioanal. Chem. 395:987–1008. Cháfer-Pericás, C., A. Maquieira, and R. Puchades.. 2010. Fast screening methods to detect antibiotic residues in food samples. Trends Analyt. Chem. 29:1038–1049. Christian, J. S. 1996. The quinolone antibiotics. Prim. Care Update Ob. Gyns. 3:87–92. De Wasch, K., L. Okerman, S. Croubels, H. De Brabander, J. Van Hoof, and P. De Backer. 1998. Detection of residues of tetracycline antibiotics in pork and chicken meat: Correlation between results of screening and confirmatory tests. Analyst 123:2737– 2741. Donkor, E., M. J. Newman, S. C. K. Tay, N. T. K. D. Dayie, E. Bannerman, and M. Olu-Taiwo. 2011. Investigation into the risk of exposure to antibiotic residues contaminating meat and egg in Ghana. Food Contr. 22:869–873. Donoghue, D. J. 2003. Antibiotic residues in poultry tissues and eggs: Human health concerns? Poult. Sci. 82:618–621. Ghosh, S., and T. M. LaPara. 2007. The effects of subtherapeutic antibiotic use in farm animals on the proliferation and persistence of antibiotic resistance among soil bacteria. ISME J. 1:191–203. Gruchalla, R. S., and M. Pirmohamed. 2006. Antibiotic allergy. N. Engl. J. Med. 354:601–609. Jafari, M. T., T. Khayamian, V. Shaer, and N. Zarei. 2007. Determination of veterinary drug residues in chicken meat using corona discharge ion mobility spectrometry. Anal. Chim. Acta 581:147–153. Juan-García, A., G. Font, and Y. Picó. 2006. Determination of quinolone residues in chicken and fish by capillary electrophoresismass spectrometry. Electrophoresis 27:2240–2249. Kaya, S., H. Yavuz, F. Akar, B. C. Liman, and A. Filazi. 1992. Mezbahadan sağlanan sığır et, karaciğer ve böbrek örneklerinde antibiyotik kalıntıları. Ankara Univ. Vet. Fak. 39:13–29. (In Turkish). Kaya, S. E., and A. Filazi. 2010. Determination of antibiotic residues in milk samples. Kafkas Univ. Vet. Fak. Derg. 16 (Suppl. A): S31–S35.
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Khadra, A., E. Pinelli, M. Z. Lacroix, A. Bousquet-Melou, H. Hamdi, G. Merlina, M. Guiresse, and M. Hafidi. 2012. Assessment of the genotoxicity of quinolone and fluoroquinolones contaminated soil with the Vicia faba micronucleus test. Ecotoxicol. Environ. Saf. 76:187–192. Koç, F. 2006. Screening of quinolone residues in animal tissues. Indian Vet. J. 83:1063–1064. Mahgoub, O., I. T. Kadim, A. Mothershaw, S. A. Al Zadjali, and K. Annamalai. 2006. Use of enzyme linked immunosorbent assay (ELISA) for detection of antibiotic and anabolic residues in goat and sheep meat. WJAS 2:298–302. Mäkinen, M., P. D. Forbes, and F. Stenback. 1997. Quinolone antibacterials: A new class of photochemical carcinogens. J. Photochem. Photobiol. B 37:182–187. Masztis, P. S. 1984. Antibiotic residue testing in a beef slaughterhouse. Can. Vet. J. 25:329–330. Moreno-Bondi, M. C., M. D. Marazuela, S. Herranz, and E. Rodriguez. 2009. An overview of sample preparation procedures for LC-MS multiclass antibiotic determination in environmental and food samples. Anal. Bioanal. Chem. 395:921–946. Naeem, M., K. Khan, and S. Rafiq. 2006. Determination of residues of quinolones in poultry products by high pressure liquid chromatography. J. Appl. Sci. Res. 6:373–379. Nisha, A. R. 2008. Antibiotic residues—A global health hazard. Vet. World. 1:375–377. Patterson, D. R. 1991. Quinolone toxicity: Methods of assessment. Am. J. Med. 91:35S–37S. Pena, A., L. J. G. Silva, A. Pereira, L. Meisel, and C. M. Lino. 2010. Determination of fluoroquinolone residues in poultry muscle in Portugal. Anal. Bioanal. Chem. 397:2615–2621. Salehzadeh, F., A. Salehzadeh, N. Rokni, R. Madani, and F. Golchinefar. 2007. Iranian Veterinary Organization (IVO), Tehran, Iran Enrofloxacin residue in chicken tissues from Tehran slaughterhouses in Iran. Pakistan J. Nutr. 6:409–413. Shareef, A. M., Z. T. Jamel, and K. M. Yonis. 2009. Detection of antibiotic residues in stored poultry products. Iraqi J. Vet. Sci. 23:45–48. Shimoda, K. 1998. Mechanism of quinolone phototoxicity. Toxicol. Lett. 102–103:369–373. Suto, M. J., J. M. Domagala, G. E. Roland, G. B. Mailloux, and M. A. Cohen. 1992. Fluoroquinolones: Relationships between structural variations, mammalian cell cytotoxicity, and antimicrobial activity. J. Med. Chem. 35:4745–4750. TFC. 2002. Turkish Food Codex, Hayvansal kökenli gıdalarda veteriner ilaçları maksimum kalıntı limitleri tebliği. Resmi Gazete: 28.04.2002–24739 Tebliğ No: 2002/30 (In Turkish). TFC. 2011. Turkish Food Codex, Hayvansal gıdalarda bulunabilecek veteriner ilaçlarına ait farmakolojik aktif maddelerin sınıflandırılması ve maksimum kalıntı limitlerinin belirlenmesi hakkında tebliğ. Resmi Gazete: 29.04.2011–27919 Tebliğ No: 2011/20 (In Turkish). Velissariou, I. M. 2006. The use of fluoroquinolones in children: Recent advances. Expert Rev. Anti Infect. Ther. 4:853–860. Vragović, N., D. Bazulic, and B. Njari. 2011. Risk assessment of streptomycin and tetracycline residues in meat and milk on Croatian market. Food Chem. Toxicol. 49:352–355. Wegener, H. 1999. The consequences for food safety of the use of fluoroquinolones in food animals. N. Engl. J. Med. 340:1581–1582. Weiss, C., A. Conte, C. Milandri, G. Scortichini, R. Semprini, G. Usberti, and G. Migliorati. 2007. Veterinary drugs residue monitoring in Italian poultry: Current strategies and possible developments. Food Contr. 18:1068–1076. WHO. 1998. Use of quinolones in food animals and potential impact on human health report of a WHO meeting. Geneva, Switzerland. Accessed Dec. 2010. http://whqlibdoc.who.int/hq/1998/ WHO_EMC_ZDI_98.10.pdf. Yüksek, N. 2001. Etlerde antibiyotik kalıntılarının aranması üzerinde çalışmalar. J. Vet. Med. Istanbul Univ. 20:85–90. (In Turkish). Zhao, S., X. Li, Y. Ra, C. Li, H. Jiang, J. Li, Z. Qu, S. Zhang, F. He, Y. Wan, C. Feng, Z. Zheng, and J. Shen. 2009. Developing and optimizing an immunoaffinity cleanup technique for determination of quinolones from chicken muscle. J. Agric. Food Chem. 57:365–371.
Among the chicken meat and beef samples, 58 (45.7%) of chicken meat samples and 60 (57.7%) of beef meat samples were positive for quinolones, respectively. The mean levels (±SE) of quinolones were found to be 30.81 ± 0.45 μg/kg and 6.64 ± 1.11 μg/kg in chicken and beef samples, respectively. This study indicated that some chicken and beef meat sold in Ankara contains residues of quinolone antibiotics.
Key words: antibiotic, chicken, enzyme-linked immunosorbent assay, quinolone, residue 2013 Poultry Science 92:2212–2215 http://dx.doi.org/10.3382/ps.2013-03072
INTRODUCTION Many antimicrobial agents are used in farm animals for therapeutic and prophylactic purposes, and as a result of this application, trace quantities of the drugs or their metabolites may be present as residues in food. The residues of these substances or their metabolites in meat and other foods of animal origin may cause adverse effects on consumers’ health (Moreno-Bondi et al., 2009). The common use of antimicrobial agents for therapeutic and nontherapeutic purposes has been mentioned as a reason for the occurrence of antibiotic resistance among human beings (Ghosh and LaPara, 2007), and thus, subtherapeutic use of these antimicrobial agents in animals is considered to have an impact for gaining resistance among environmental bacteria (Bester and Essack, 2010). Therefore, the analysis of the residues of these compounds in meat and meat products is very important for human health (Mahgoub et al., 2006). In recent years, a public health consequence of the use of antibiotics in food animals has grown (Jafari et al., 2007). Foodborne bacterial pathogens are resistant to antibiotics, resulting in increased human morbidity and mortality (Wegener, 1999).
©2013 Poultry Science Association Inc. Received January 28, 2013. Accepted May 10, 2013. 1 Corresponding author: [email protected]
Quinolones and fluoroquinolones are important antibiotics used in human and veterinary medicine (Velissariou, 2006; Andreu et al., 2007; Cañada-Cañada et al., 2009; Cháfer-Pericás et al., 2010). Quinolones may have a directly toxic effect or lead to the emergence of drug-resistant bacteria predicating a potential risk to human health. These residues can produce allergic hypersensitivity reactions or toxic effects (Juan-García et al., 2006; Cañada-Cañada et al., 2009). Presently, several quinolones are acceptable for treatment of beef, poultry, and fish in many countries of the world (WHO, 1998). In poultry, antibiotics are used for reducing the incidence of disease and for growth promotion. Antibiotics promote growth by decreasing activity of the immune system and reducing waste of nutrients and toxin formation. Unfortunately, contaminated poultry tissues with harmful concentrations of drug residues may occur (Donoghue, 2003; Nisha, 2008; Shareef et al., 2009). Quinolone is one of the most widely used antibiotics in veterinary medicine for treatment and prevention of diseases. Residues of quinolone may be observed in foods of animal origin if proper checks and balances are not employed. It has been reported that, because of the presence of quinolone residues in human food obtained from animal sources, effectiveness of quinolones in human treatment has decreased (Donkor et al., 2011). In Turkey, ciprofloxacin, enrofloxacin, marbofloxacin, danofloxacin, difloxacin, sarafloxacin, flumequin, and oxolinic acid are approved quinolones for usage in ani-
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SCREENING OF QUINOLONE ANTIBIOTIC RESIDUES
mal production. Among these quinolones, sarafloxacin is permitted in skin, wing meat, and liver of chicken but not in breast meat. According to the Turkish Food Codex (TFC), the maximum residue levels of the sum of enrofloxacin and ciprofloxacin in chicken muscle and beef muscle should not exceed 100 µg/kg; marbofloxacin should not exceed 150 µg/kg in beef muscle; danofloxacin should not exceed 200 µg/kg in chicken muscle and beef muscle; difloxacin should not exceed 300 µg/ kg in chicken muscle and 400 µg/kg in beef muscle; flumequin should not exceed 400 µg/kg in chicken muscle and 200 µg/kg in beef muscle; and oxolinic acid should not exceed 100 µg/kg in food animal muscle (TFC, 2002, 2011). The aim of this study was to determine the quinolone residues in chicken meat and beef samples collected from Ankara region local markets.
MATERIALS AND METHODS Sample Collection In this study, 127 chicken breast meat and 104 beef round meat samples were collected from local markets in 3 different districts (A, B, C) of Ankara, Turkey. Between July 2010 and September 2010, chicken meats were collected, and between September 2010 and October 2010, beef meats were collected. Among beef round samples, 50 samples originated from domestic animals and 54 samples originated from foreign animals. The samples were preserved at 4°C soon after collection. Methanol was purchased from Merck (Darmstadt, Germany).
Analysis of Samples Quinolone residues were determined with an ELISA using the Ridascreen Quinolones/ELISA kit (RBiopharm AG, Darmstadt, Germany). The assay was performed according to the guidelines of the manufacturers. The ELISA kit showed cross-reactivity with ciprofloxacin (100%), norfloxacin, enrofloxacin, marbofloxacin, danofloxacin, difloxacin, flumequin, ofloxacin (>100%), sarafloxacin (43%), and oxolinic acid (24%). The detection limit was reported as 5 µg/kg, being the lowest value for chicken meat and beef in the test kit. Sample preparations were done according to the instructions of the Ridascreen kit. Briefly, each of randomly selected muscle tissue sample was homogenized using a Turrax disperser and 5 g of the homogenized sample was transferred to centrifuge tubes, containing 4 mL of methanol/water (70:30, vol/vol) and vortexed for 10 min. Subsequently, this was centrifuged at 4,000 × g for 10 min. Fifty microliters of the standard solution and each of the samples were added to 96-well microplates. Fifty microliters of enzyme conjugate and 50 µL of antibody were added to the wells, respectively, and mixed gently and incubated for 1 h at 4°C. At the end of the incubation the liquid was poured from the wells and washed twice with the washing buffer. One
2213
hundred microliters of the substrate/chromogen was added to the wells and incubated at room temperature for 15 min. One hundred microliters of the stop solution was added to each well and the absorbance was measured at 450 nm in ELISA plate reader (VersaMax Tunable). Quinolones concentrations were calculated through the guidelines of the Ridascreen kit (R-Biopharm AG). Calibration curves were plotted as semilogarithmic concentration versus the ratio of the mean absorbance at each concentration divided by the mean absorbance of the zero standard (B/Bo). The B/Bo values were decreasing, which is a good result for the reliability of the test. The results of the samples were calculated after the B/Bo values were evaluated.
Statistical Analyses Independent sample t-test and Shapiro-Wilk test were conducted for statistical comparison.
RESULTS AND DISCUSSION One hundred eighteen of 231 (51.1%) examined chicken meat and beef samples were found to contain quinolone antibiotic residue. Fifty-eight (45.7%) of the chicken meat samples were positive for quinolones, and 60 (57.7%) of beef meat samples were positive for quinolones. The mean levels of quinolone antibiotic residue were found to be 30.81 ± 0.45 µg/kg in positive chicken samples. However, quinolone residues were found in low levels in beef samples. The mean levels of quinolone residues were as low as 6.64 ± 1.11 µg/kg in positive beef samples. Our data revealed that quinolone mean levels in chicken and beef meat samples were lower than 100 µg/kg, which is the lowest residue value mentioned in the TFC. This study indicated that some chicken and beef meat sold in Ankara contains residues of quinolone antibiotic, although these levels were low. In 2 of chicken samples, a level of quinolone was determined to be higher than 100 µg/kg. These levels were determined as 103 and 106.2 µg/kg. The concerning values are shown in Tables 1 and 2. Limited studies on antibiotic residues in poultry animals have been performed in Turkey. However, considering the importance of antibiotic residues in foodstuffs, periodic sampling is carried out in many countries (Salehzadeh et al., 2007; Weiss et al., 2007; Zhao et al., 2009; Pena et al., 2010). Akar (1994) reported 175 chicken meat samples in Ankara and found antibiotic (chloramphenicol, erythromycin, and tylosin) residue rate to be 5.7%. Yüksek (2001) reported 50 chicken meat samples in Ankara and did not find residue of oxytetracycline, chloramphenicol, and zinc bacitracin in muscle tissues of the chicken. Pena et al. (2010) analyzed 98 samples of chicken samples in the north and central zones of Portugal and found fluoroquinolones residue rates to be 44.2%. Zhao
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Er et al. Table 1. Statistical analyses for levels of quinolone in positive samples of beef meat1 Origin of beef meat
Sample tested, n
Positive samples, n (%)
Concentration of positive samples, µg/kg (mean ± SE)
Foreign Domestic Total
54 50 104
33 (61.1) 27 (54.0) 60 (57.7)
6.74 ± 0.21 6.50 ± 0.20 6.64 ± 0.14
1There is no statistically significant (P > 0.05) difference between foreign and domestic beef meats in case of quinolone concentrations.
et al. (2009) determined 11.7% quinolone residue rate in 14 of 120 chicken muscle samples and found quinolone residue levels of 0.7 to 43.6 ng/g. Salehzadeh et al. (2007) determined 270 chicken muscle, liver, and kidney samples from 90 broiler farms in Tehran, Iran, and found enrofloxacin mean concentrations as 18.32 ± 32.29, 18.34 ± 12.36, and 26.06 ± 19.52 ng/g in muscle, liver, and kidney samples, respectively. Weiss et al. (2007) analyzed 299 samples of poultry meat in Italy and could not found enrofloxacin antibiotic residue. These contamination rates in chicken samples were lower in comparison with values reported in this study. The literature available on the occurrence of antibiotics in poultry samples indicates higher rates of antibiotic residues. Shareef et al. (2009) reported antibiotic residue rate as 52% in poultry products from a total of 75 samples. Naeem et al. (2006) analyzed poultry products in Pakistan and reported that 58 to 85% of the samples contained ciprofloxacin residues and 55 to 92% of the samples contained enrofloxacin residues in violation of the regulations. In Turkey, there is limited number of studies related to the search for antibiotics in cattle. Kaya et al. (1992) reported the antibiotic residue rate as 18% in samples from a total of 85 cattle in Turkey. Koç (2006) analyzed 240 samples of cattle and sheep meats in Ankara, Turkey, and did not find quinolone antibiotic residue. These results in cattle samples show compatibility with the results of this study. Quinolone antibiotic residues contamination is also reported in milk samples collected from Turkey. Kaya and Filazi (2010) determined 1.25% antibiotic residue contamination rates in milk samples. Alla et al. (2011) analyzed beef samples in Sudan and reported that 3% of the muscles contain antibiotic residue. Donkor et al. (2011) reported antibiotic residue rate of 30.8% in samples from a total of 156
beef samples in Ghana. Masztis (1984) analyzed 487 bovine carcasses in Canada and found that 12 samples were positive. Antibiotic residues can be found at different concentration levels in foods of animal origin such as fish, beef, poultry, pork, milk, and milk products (De Wasch et al., 1998; Vragović et al., 2011). Consequently, antibiotic residues have been indicated as a public health concern. Quinolones are potent inhibitors of DNA gyrase enzyme, which is critical for DNA replication and transcription (Suto et al., 1992). The toxicity and side effects of quinolones are well established in animals and humans (Christian, 1996; Mäkinen et al., 1997; Shimoda, 1998; Khadra et al., 2012). Fluoroquinolones show damage to the juvenile joint, the kidney, the eye, and the central nervous system, which has been previously reported by animal experiments (Patterson, 1991). Some antibiotic-induced allergic reactions have also been reported, in relation to quinolones (Gruchalla and Pirmohamed, 2006). Given the adverse effects of quinolones on human health, residues in food and other environmental sources are also expected to affect human health. Therefore, it is necessary to monitor frequently consumed meat and meat products with high nutritional value for the presence of residual quinolones. The analysis of residues of these drugs in edible tissues is very important for human health. In Turkey, using quinolone for the prophylaxis and treatment of chicken and beef is legal, but there is a limit in the preslaughter withdrawal times. Also, there is a limit for the quinolone residues in chicken and beef meat in the TFC. According to these limitations, low levels of quinolone residues in chicken and beef meat is not a surprising but a good result. These results are valid for the obtained samples during the study. Results may change periodically. Therefore,
Table 2. Statistical analyses for levels of quinolone in positive samples of chicken meat
District A B C Total
Samples tested, n 41 56 30 127
Positive samples, n (%) 12 37 9 58
(29.3) (66.1) (30.0) (45.7)
Concentration of positive samples, µg/kg (mean ± SE) 15.23 36.65 27.58 30.81
± ± ± ±
2.22 4.04 7.30 3.04
Mean value of samples 14.03a 36.03b 16.33a
a,bFor an attribute, means within a groups in a column (between different districts) not having a common superscript letter are different (P < 0.05).
SCREENING OF QUINOLONE ANTIBIOTIC RESIDUES
the steps in food processing should be kept under continuous monitoring for preventing overexpressed levels of quinolone residues. In conclusion, the chicken and beef samples obtained during the period of the study are thought to exclude health risks in terms of quinolone presence. In terms of preventing antibiotic resistance in humans, the low amount of quinolone residue levels observed in this study represents a positive result for local food control.
ACKNOWLEDGMENTS This study was partly supported by Gazi University Research Foundation (project no. 02/2010-24). The authors are thankful to Khalid Mahmood Khawar, Department of Field Crops, Faculty of Agriculture, Ankara University, for guidance and preparation of the manuscript.
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