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Residues of tetracycline compounds in poultry products in the eastern province of Saudi Arabia
Public Health (2000) 114, 300±304 ß R.I.P.H.H. 2000 www.nature.com/ph
Residues of tetracycline compounds in poultry products in the eastern province of Saudi Arabia MS Al-Ghamdi1*, ZH Al-Mustafa1, F El-Morsy2, A Al-Faky1, I Haider1 and H Essa2 Departments of 1Pharmacology and 2Microbiology, College of Medicine, King Faisal University, Dammam, Saudi Arabia Chicken muscle, liver and egg samples were collected from 33 broiler and 5 layer farms in the eastern province of Saudi Arabia over a period of two years starting from January 1996. Antibiotic-residue positive samples were identi®ed in the products of 23 (69.7%) broiler and 3 (60%) layer poultry farms. 87% and 100.0% of the antibiotic-residue positive broiler farms were positive for at least one tetracycline compound in raw muscle and liver respectively, while 73.9% and 95.5% were positive for 2 or more tetracyclines in these two tissues, respectively. Furthermore, 82.6% and 95.5% of the antibiotic-residue-positive farms had mean concentrations of at least one tetracycline compound in excess of the permissible maximum residue limit (MRL) in raw muscle and liver, respectively. These compounds also remained chemically detectable after cooking. Tetracycline levels exceeded MRL in 14.4% of antibiotic-positive raw eggs but the overall mean tetracycline concentration in each farm was below MRL. This study con®rmed widespread misuse of tetracycline agents including multiple use of drugs belonging to the same pharmacological group and lack of implementation of recommended withdrawal times. This may be contributing to the high resistance rates to tetracyclines in both chicken and human microbial isolates observed in the region. This study, therefore, stresses the need for stricter regulations for the use of antimicrobial drugs in the poultry industry as well as the inspection of chicken for drug residues prior to marketing. Public Health (2000) 114, 300±304. Keywords: antibiotics; tetracycline; residue; poultry; Saudi Arabia
Introduction Tetracycline agents are of great clinical importance because they possess a wide range of antimicrobial activity against aerobic and anaerobic gram-positive and gramnegative bacteria. They are also effective against some microorganisms that are resistant to cell-wall-inhibitor antimicrobial agents, such as Rickettsia, Mycoplasma pneumoniae, Chalamydia spp., Legionella spp., Ureaplasma, some atypical mycobacteria and Plasmodium spp.1 However, resistance to tetracyclines is common and microorganisms that have become resistant to one tetracycline compound frequently exhibit resistance to others.1 The use of antimicrobial agents in food-producing animals has recently become a very important public health issue. This is due to the fact that these agents are being increasingly used in farm animal production. Many antimicrobial agents are routinely added to animal feed at sub-therapeutic levels for their growth promoting properties.2 In addition, antimicrobials are widely used for disease prophylaxis and treatment, an important measure when raising animals under intensive husbandry methods of production.3 ± 5 Thus, these agents are considered very valuable in preventing major economic losses to the grower caused by disease outbreaks. This practice, however, carries many disadvantages, such as the stimulation of microbial resistance and the presence of drug residues in animal products which may pose a major health risk to the public.5 ± 10 Internationally recognized organizations such as World Health Organization (WHO), Food and Agriculture Organization (FAO),11 Veterinary Medicines Directorate (VMD)12 of the European Union as well as Food and *Correspondence: Dr Mastour Al-Ghamdi, Department of Pharmacology, King Faisal University, College of Medicine, PO Box 2114, Dammam 31451, Saudi Arabia. Tel: ( 966) 3-8577000 X2238; Fax: ( 966) 3- 8575329. Accepted 11 February 2000
Drug Administration (FDA)13 of the USA have set tolerance or maximum residue limits (MRLs), acceptable daily intakes (ADIs) for humans and withholding times for pharmacologically active substances including antimicrobial agents prior to marketing. In the eastern province of Saudi Arabia, 29 antimicrobial agents are available for poultry use (own survey). However, due to their relatively low cost, tetracycline compounds are the most commonly dispensed by poultry producers. This study was therefore aimed at identifying the tetracycline compounds and determining their levels in edible chicken products (muscle, liver and eggs) ready for marketing. Methods 247 chicken-muscle, 719 chicken-liver and 630 eggs samples were collected over 49 inspection visits to 33 broiler and 5 layer farms in the eastern province of Saudi Arabia over a period of two years, starting from January 1996. All samples were screened for antibiotic residues using microbiological methods as follows. Single solid pieces (50 ± 100 mg) of raw muscle and liver were transferred into wells in agar plates (Mueller Hinton, Oxiod, England) under sterile conditions. Similarly, 100 ml of raw egg were pipetted into the agar plates using sterile adjustable micropipettes. The agar plates were previously seeded with reference strains of Staphylococcus aureus (ATCC 25923), Escherichia coli (ATCC 35218), Pseudomonas aeruginosa (ATCC 27853) and Bacillus subtillis (B.B.L 6633). The plates were then incubated at 37 C for 20 h. These tests were performed in duplicate and the presence of inhibition zones in any sample was considered to indicate the presence of antibiotic residues. In such cases further analysis by high performance liquid chromatography (HPLC) for tetracycline drugs (oxytetracycline (OXT), tetracycline (TET), chlortetracycline (CHT) and doxycycline (DXC)) was carried out as follows.
Residues of tetracycline compounds in poultry products MS Al-Ghamdi et al
Soup was prepared by cooking pieces of muscle and liver (10 ± 20 g) in 5 volumes of water at 100 C for 20 min. Homogenates of both raw and cooked tissues were then prepared in 10 volumes of 2.0 M sodium phosphate= sulphite buffer (pH 6.1). Raw and cooked tissue homogenates, soup and raw egg were extracted and assayed by HPLC for the concentrations of tetracycline agents as previously described by Gastearena et al.14 Results Antibiotic-residue positive samples were identi®ed in 23 (69.7%) out of the 33 broiler and 3 (60%) out of the 5 layer poultry farms investigated. The results of 110 raw liver samples as well as 117 raw chicken-muscle samples obtained from 22 and 23 broiler farms, respectively (liver samples from one farm were lost), indicate that one or more compounds of tetracyclines were used in these farms. Tables 1 and 2 summarize the concentrations of tetracycline compounds in raw and cooked liver respec-
tively. OXT was detectable in 90.9% of the farms and 77.3% of raw chicken-liver samples (Table 1). After cooking, the overall mean concentration of this drug increased by 45% and it was also found in a greater percentage of farms and samples (Table 2). TET, CHT and DXC were also positively detected in 46.4%, 53.6% and 33.6% of the investigated raw samples, respectively. However, unlike OXT, their concentration as well as the number of positive samples decreased after cooking. A similar pattern of results was also observed in muscle samples, with OXT having the highest concentration and being the most frequently detected (Tables 3 and 4). Table 5 summarizes the drug concentrations in the soup of cooked chicken tissue (muscle and liver). These results again show that all four investigated tetracycline agents, especially OXT, were detectable in appreciable concentrations in soup samples. Mean concentrations of OXT in raw and cooked liver were above the liver maximum residue limit (MRL) (0.30 mg=g) in the majority of residue Ð positive farms (Figure 1). In addition, the MRL for TET, CHT and DXC
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Table 1 Summary of tetracycline drug residues in 110 raw chicken livers obtained from 22 farms
Range of farm mean conc. (mg=g) Overall mean conc. (mg=g) SD % positive farms % positive sample
OXT
TET
CHT
DXC
0.15 ± 2.93 1.00 0.78 90.9 77.3
0.15 ± 2.89 0.84 0.84 50.0 46.4
0.12 ± 3.24 0.88 0.87 59.1 53.6
0.17 ± 1.13 0.36 0.87 40.9 33.6
OXT: oxytetracycline, TET: tetracycline, CHT: chlortetracycline, DXC: doxycycline. Table 2 Summary of tetracycline drug residues in 110 cooked chicken livers obtained from 22 farms
Range of farm mean conc. (mg=g) Overall mean conc. (mg=g) SD % positive farms % positive sample
OXT
TET
CHT
DXC
0.17 ± 4.38 1.45 1.14 100 90.9
0.04 ± 0.20 0.13 0.08 18.2 12.7
0.13 ± 1.05 0.46 0.36 50.0 37.3
0.15 ± 0.20 0.18 0.02 27.3 19.1
OXT: oxytetracycline, TET: tetracycline, CHT: chlortetracycline, DXC: doxycycline. Table 3 Summary of tetracycline drug residues in 117 raw chicken muscles obtained from 23 farms
Range of farm mean conc. (mg=g) Overall mean conc. (mg=g) SD % positive farms % positive sample
OXT
TET
CHT
DXC
0.23 ± 2.05 0.96 0.54 78.3 62.4
0.11 ± 3.37 0.72 0.93 47.8 43.6
0.11 ± 2.92 0.82 0.93 43.5 38.5
0.18 ± 1.20 0.77 0.40 21.7 16.2
OXT: oxytetracycline, TET: tetracycline, CHT: chlortetracycline, DXC: doxycycline. Table 4 Summary of tetracycline drug residues in 117 cooked chicken muscles obtained from 23 farms
Range of farm mean conc. (mg=g) Overall mean conc. (mg=g) SD % positive farms % positive sample
OXT
TET
CHT
DXC
0.12 ± 2.35 1.02 0.70 73.9 58.1
0.06 ± 0.32 0.19 0.11 26.1 20.5
0.10 ± 0.93 0.43 0.30 30.4 25.6
0.20 ± 0.22 0.21 0.01 8.7 7.7
OXT: oxytetracycline, TET: tetracycline, CHT: chlortetracycline, DXC: doxycycline. Public Health
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Table 5 Summary of tetracycline drug residues in cooking ¯uid (soup) of 110 chicken muscle and liver obtained from 22 farms
Range of farm mean conc. (mg=g) Overall mean conc. (mg=g) SD % positive farms % positive sample
OXT
TET
CHT
DXC
0.08 ± 0.67 0.24 0.16 100 86.4
0.06 ± 0.30 0.15 0.09 31.8 28.2
0.04 ± 0.24 0.10 0.06 50.0 43.6
0.03 ± 0.23 0.09 0.07 31.8 23.6
OXT: oxytetracycline, TET: tetracycline, CHT: chlortetracycline, DXC: doxycycline.
Figure 1 Mean concentrations of oxytetracycline in raw and cooked chicken liver. The maximum residue limit (MRL) 0.30 mg=g.
Figure 2 Mean concentrations of oxytetracycline in raw and cooked chicken muscle. The maximum residue limit (MRL) 0.10 mg=g.
was also exceeded in raw liver samples in 9, 9 and 3 farms, respectively. However, after cooking the mean detectable concentrations of these three drugs were decreased to below MRL except for CHT in 5 farms. Mean concentrations of OXT in raw muscle exceeded the muscle MRL value (0.10 mg=g) in all 18 OXT Ð positive farms and they remained above the MRL in most farms even after cooking (Figure 2). Furthermore, mean concentrations of TET, CHT and DXC were also above the MRL in raw muscle obtained from 10, 9 and 5 farms, respectively. However, after cooking, the MRL of these drugs was exceeded only in 3, 6 and 2 of the farms, respectively. One tetracycline agent, namely TET, was also detected in 180 raw eggs collected from 3 farms. Mean levels ranged from 0.08 ± 0.25 mg=g (overall farm mean s.d. 0.15 0.04). Although 14.4% of the investigated egg samples exceeded the egg MRL value (0.20 mg=g), mean concentrations of TET in all farms were below MRL.
with the known pharmacokinetic pro®les of these drugs. Tetracyclines distribute widely into body tissues and are found in high concentrations in the excretory organs, especially the liver and in the bile. Tetracyclines undergo extensive entrohepatic circulation which leads to prolongation of their elimination half-lives; thus persisting in the body for a long time after cessation of drug administration.15 Long withholding periods for oral use of tetracyclines (up to 21 d depending on indications and type of drug) have therefore been recommended in the USA.16 OXT had the highest concentration and was the most frequently detected drug in all types of samples investigated. This may be due to the fact that long-term administration of OXT in feed is particularly recommended for controlling Mycoplasma gallispeticum infection which is considered to be one of the most common causes of chicken morbidity.15,17 However, as a feed additive the maximum recommended amount of this drug is 100 ± 200 g=ton, depending on indications, either nutritional or prophylactic.17 For economic reasons, local poultry producers add this drug to water, a practice that may enhance the absorption of the drug and ultimately its tissue concentration. The MRLs for tetracycline compounds in all foodproducing animals have been set at 0.10, 0.30 and 0.20 mg=g in muscle, liver and eggs, respectively, while
Discussion In the present study, high levels of tetracycline compounds were detected in edible chicken tissue, with liver having higher concentrations than muscle. This is in agreement Public Health
Residues of tetracycline compounds in poultry products MS Al-Ghamdi et al
ADI for humans is recommended not to exceed 3.0 mg.11,12,18,19 The results of the present study show that the overall farm mean concentrations of various tetracyclines in raw and cooked muscle were 1.9 ± 10 times greater than the recommended MRL value. Similarly, for raw liver, the overall farm means for these agents exceeded the recommended MRL by 1.2 ± 3.3 times and only the mean concentrations of TET and DXC fell below MRL following cooking. In addition, the results also show that the overwhelming majority of investigated farms (82.6% and 95.5%) had mean concentrations of at least one tetracycline compound in raw muscle and liver, respectively, in excess of MRL. Furthermore, 36.4% and 13.4% of all collected muscle and liver samples respectively had tetracycline drug levels that were above the MRL. In comparison, the percentage of residue violations for tetracyclines detected in 1777 chickens in the USA under the monitoring program mounted by the United States Department of Agriculture's Food Safety and Inspection Service (USDA=FSIS) between 1983 and 1990 was only 0.06%.16 It is currently recognized that the incidence of residue violations should not exceed 1.0% of samples and that levels above it indicate improper use of antimicrobial agents, especially the failure to adhere to recommended withdrawal times.16 The effect of cooking on antibiotic residues was also investigated in the same samples. The results showed that the overall mean levels of TET, CHT and DXC in both muscle and liver decreased markedly after cooking. On the other hand, the overall mean levels of OXT in cooked tissues were higher than in raw ones but the difference does not reach statistical signi®cance (P > 0.05). This may re¯ect the high stability of OXT to heating. It has been documented that OXT crystals remain potent after heating for 4 d at 100 C.20 However, the overall farm mean levels of all four agents in cooked muscle remained above the internationally recommended MRL while only those of TET and DXC fell below MRL in cooked liver. Extensive use of multiple tetracycline agents in broiler farms was also detected in this study with 73.9% and 95.5% of investigated farms testing positive for 2 or more tetracyclines in raw muscle and liver, respectively. While OXT and CHT have been approved in the USA for nutritional, prophylactic and therapeutic uses in poultry, TET and DXC are to be used only as therapeutic agents.17 In the present study, all four tetracyclines were detectable at the time of slaughter, indicating that the four agents were being continuously used throughout the production cycle. Multi-tetracycline use is not clinically justi®able as these drugs share the same mechanism of action and cover the same microbial spectrum. This practice may contribute signi®cantly to the development of microbial resistance to the drugs. Indeed, our own results (unpublished data) showed that 89.2% of 1962 chicken normal ¯ora isolates were resistant to tetracyclines, while on the other hand 52.8% of human pathogenic organisms, investigated for tetracyclines susceptibility in a referral hospital in the region, were resistant to this drug. Many reports indicate that microbial resistance to antibiotics may arise as a result of animal exposure to antimicrobial agents with the resistance being possibly transferred to human pathogens.5,7,8,21 ± 23 In addition, human exposure to animal products containing signi®cant levels of antibiotic residues may provoke immunological responses in susceptible individuals9 and cause disorders of intestinal ¯ora.8,10
Only one tetracycline agent (TET) was detected in eggs and with mean levels in all 3 positive farms below the recommended MRL. However, this may be due to growers' awareness of the impact of tetracycline administration on calcium absorption which is essential for egg production. In conclusion, the results of this study indicate that there is widespread misuse of tetracycline agents by poultry producers in the eastern province of Saudi Arabia, possibly re¯ecting a general lack of compliance with internationally recommended practices regarding the use of antimicrobial agents. This malpractice includes multiple use of drugs belonging to the same pharmacological group and lack of implementation of recommended withdrawal times. It stresses the need for stricter regulations for the use of antimicrobial drugs in the poultry industry as well as inspection of chicken for drug residues prior to marketing.
303
Acknowledgements This work was funded by the King Abdulaziz City of Science and Technology (KACST), Saudi Arabia (Grant no. AT-15-79) to whom we express our gratitude. Our sincere thanks also go to Dr Abdul-Rahman Qurashi, and the technical staff at the Microbiology laboratories at King Fahd Hospital of the University and College of Medicine for their assistance.
References 1 Kapusnik-Uner JE, Sande MA, Chambers HF. Tetracyclines, chloramphenicol, erythromycin and miscellaneous antibacterial agents. In: Hardman JG, Limbird LE, Molinoff PB, Ruddon RW, Gilman AG (eds.), Goodman and Gilman's the Pharmacological Basis of Therapeutics, 9th edn. McGrawHill: New York, 1996, pp. 1123 ± 1153. 2 Droumev D. Review of antimicrobial growth promoting agents available. Vet Res Commun 1983; 7: 85 ± 99. 3 Swan Report. Report of the Joint Committee on the Use of Antibiotics in Animal Husbandry and Veterinary Medicine. cmnd 4190, HMSO: London, 1969. 4 Smith HW. Veterinary and food aspects of drug resistance. J Sci Food Agricult 1974; 25: 227 ± 237. 5 Aronson AL. Potential impact of the use of antimicrobial drugs in farm animals on public health. Presented at the Meeting on Pharmacology in the Animal Health Sector. Colorado State University, Fort Collins, 1975. 6 Linton AH. Antibiotics, animals and man. An appraisal of a continuous subject. In: Woobine M (ed.), Antibiotics and Antibiosis in Agriculture, 1st edn. Butterworths: London, 1977, pp. 315 ± 343. 7 Mercer HD. Antimicrobial drugs in food producing animals. Vet Clin North Am 1977; 5: 3 ± 5. 8 Holmberg SD, Osterholm MT, Senger KA, Cohen ML. Drugresistant Salmonella from animals fed antimicrobials. New Engl J Med 1984; 311: 617 ± 622. 9 Woodward KN. Hypersensitivity in humans and exposure to veterinary drugs. Vet Hum Toxicol 1991; 33: 168 ± 172. 10 Archimbault PH. Persistence in milk of active antimicrobial intramammary substances. In: Ruckebusch YR, Toutain PL, Kortiz GD (eds.), Veterinary Pharmacology and Toxicology. MTP Press Ltd: Falkon House, Lancaster, 1983. 11 FAO=WHO. 32nd Report of the Joint FAO=WHO Expert Committee on Food Additives. Evaluation of Certain Veterinary Drug Residues in Food. WHO=FAO Tech Rep Ser 1988; 763:1. Public Health
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12 Veterinary Medicines Directorate (VMD). List of pharmacologically active substances for which maximum residue limits have been ®xed. Regulation 2377=90, Annex I, VMD, European Commission, Brussels, 22 October 1997. 13 Food and Drug Administration. Compliance Policy Guide 7126.06.1986. Extra-Label Use of New Animal Drugs in Food-Producing Animals. Food and Drug Administration, Rockville, MD, USA, 1986. 14 Gastearena MC et al. Determination of doxycycline in small serum samples by liquid chromatography. Chromatographia 1993; 35: 524 ± 526. 15 Prescott JF, Baggot JD. Tetracyclines. In: Prescott JF, Baggot JD (eds.), Antimicrobial Therapy in Veterinary Medicine, 2nd edn. Iowa State University Press: Iowa, 1993, pp. 215 ± 228. 16 Sundlof SF. Antimicrobial drug residues in food-producing animals. In: Prescott JF, Baggot JD (eds.), Antimicrobial Therapy in Veterinary Medicine, 2nd edn. Iowa State University Press: Iowa, 1993, pp. 569 ± 591. 17 Tanner AC. Antimicrobial drug use in poultry. In: Prescott JF, Baggot JD (eds.), Antimicrobial Therapy in Veterinary Medicine, 2nd edn. Iowa State University Press: Iowa, 1993, pp. 507 ± 523.
Public Health
18 FAO=WHO. 34th Report of the Joint FAO=WHO Expert Comittee on Food Additives. Evaluation of Certain Veterinary Drug Residues in Food. WHO=FAO Tech Rep Ser 1989; 788:1. 19 FAO=WHO. 36th Report of the Joint FAO=WHO Expert Committee on Food Additives. Evaluation of Certain Veterinary Drug Residues in Food. WHO=FAO Tech Rep Ser 1990; 799:1. 20 Windholz M, Budavari S, Stoumtsos LY, Fertig MN. Merck Index: An Encyclopedia of Chemicals and Drugs, 9th edn. Merck & Co. Inc.: Rahaway, NJ, 1976, p. 6790. 21 Sogaard H. Incidence of drug resistance and transmissible R factors in strains of E. coli isolated from faeces of healthy pigs. Acta Vet Scand 1973; 14: 381 ± 391. 22 Anderson ES. Drug resistance in Salmonella typhimurium and its implications. Br Med J 1968; 10: 333 ± 339. 23 Al-Ghamdi MS, El-Morsy F, Al- Mustafa ZH, Al-Ramadhan M, Hanif M. Antibiotic resistance of Escherichia coli isolated from poultry workers, patients and chicken in the eastern province of Saudi Arabia. Trop Med Int Health 1999; 4: 278 ± 283.
Residues of tetracycline compounds in poultry products in the eastern province of Saudi Arabia MS Al-Ghamdi1*, ZH Al-Mustafa1, F El-Morsy2, A Al-Faky1, I Haider1 and H Essa2 Departments of 1Pharmacology and 2Microbiology, College of Medicine, King Faisal University, Dammam, Saudi Arabia Chicken muscle, liver and egg samples were collected from 33 broiler and 5 layer farms in the eastern province of Saudi Arabia over a period of two years starting from January 1996. Antibiotic-residue positive samples were identi®ed in the products of 23 (69.7%) broiler and 3 (60%) layer poultry farms. 87% and 100.0% of the antibiotic-residue positive broiler farms were positive for at least one tetracycline compound in raw muscle and liver respectively, while 73.9% and 95.5% were positive for 2 or more tetracyclines in these two tissues, respectively. Furthermore, 82.6% and 95.5% of the antibiotic-residue-positive farms had mean concentrations of at least one tetracycline compound in excess of the permissible maximum residue limit (MRL) in raw muscle and liver, respectively. These compounds also remained chemically detectable after cooking. Tetracycline levels exceeded MRL in 14.4% of antibiotic-positive raw eggs but the overall mean tetracycline concentration in each farm was below MRL. This study con®rmed widespread misuse of tetracycline agents including multiple use of drugs belonging to the same pharmacological group and lack of implementation of recommended withdrawal times. This may be contributing to the high resistance rates to tetracyclines in both chicken and human microbial isolates observed in the region. This study, therefore, stresses the need for stricter regulations for the use of antimicrobial drugs in the poultry industry as well as the inspection of chicken for drug residues prior to marketing. Public Health (2000) 114, 300±304. Keywords: antibiotics; tetracycline; residue; poultry; Saudi Arabia
Introduction Tetracycline agents are of great clinical importance because they possess a wide range of antimicrobial activity against aerobic and anaerobic gram-positive and gramnegative bacteria. They are also effective against some microorganisms that are resistant to cell-wall-inhibitor antimicrobial agents, such as Rickettsia, Mycoplasma pneumoniae, Chalamydia spp., Legionella spp., Ureaplasma, some atypical mycobacteria and Plasmodium spp.1 However, resistance to tetracyclines is common and microorganisms that have become resistant to one tetracycline compound frequently exhibit resistance to others.1 The use of antimicrobial agents in food-producing animals has recently become a very important public health issue. This is due to the fact that these agents are being increasingly used in farm animal production. Many antimicrobial agents are routinely added to animal feed at sub-therapeutic levels for their growth promoting properties.2 In addition, antimicrobials are widely used for disease prophylaxis and treatment, an important measure when raising animals under intensive husbandry methods of production.3 ± 5 Thus, these agents are considered very valuable in preventing major economic losses to the grower caused by disease outbreaks. This practice, however, carries many disadvantages, such as the stimulation of microbial resistance and the presence of drug residues in animal products which may pose a major health risk to the public.5 ± 10 Internationally recognized organizations such as World Health Organization (WHO), Food and Agriculture Organization (FAO),11 Veterinary Medicines Directorate (VMD)12 of the European Union as well as Food and *Correspondence: Dr Mastour Al-Ghamdi, Department of Pharmacology, King Faisal University, College of Medicine, PO Box 2114, Dammam 31451, Saudi Arabia. Tel: ( 966) 3-8577000 X2238; Fax: ( 966) 3- 8575329. Accepted 11 February 2000
Drug Administration (FDA)13 of the USA have set tolerance or maximum residue limits (MRLs), acceptable daily intakes (ADIs) for humans and withholding times for pharmacologically active substances including antimicrobial agents prior to marketing. In the eastern province of Saudi Arabia, 29 antimicrobial agents are available for poultry use (own survey). However, due to their relatively low cost, tetracycline compounds are the most commonly dispensed by poultry producers. This study was therefore aimed at identifying the tetracycline compounds and determining their levels in edible chicken products (muscle, liver and eggs) ready for marketing. Methods 247 chicken-muscle, 719 chicken-liver and 630 eggs samples were collected over 49 inspection visits to 33 broiler and 5 layer farms in the eastern province of Saudi Arabia over a period of two years, starting from January 1996. All samples were screened for antibiotic residues using microbiological methods as follows. Single solid pieces (50 ± 100 mg) of raw muscle and liver were transferred into wells in agar plates (Mueller Hinton, Oxiod, England) under sterile conditions. Similarly, 100 ml of raw egg were pipetted into the agar plates using sterile adjustable micropipettes. The agar plates were previously seeded with reference strains of Staphylococcus aureus (ATCC 25923), Escherichia coli (ATCC 35218), Pseudomonas aeruginosa (ATCC 27853) and Bacillus subtillis (B.B.L 6633). The plates were then incubated at 37 C for 20 h. These tests were performed in duplicate and the presence of inhibition zones in any sample was considered to indicate the presence of antibiotic residues. In such cases further analysis by high performance liquid chromatography (HPLC) for tetracycline drugs (oxytetracycline (OXT), tetracycline (TET), chlortetracycline (CHT) and doxycycline (DXC)) was carried out as follows.
Residues of tetracycline compounds in poultry products MS Al-Ghamdi et al
Soup was prepared by cooking pieces of muscle and liver (10 ± 20 g) in 5 volumes of water at 100 C for 20 min. Homogenates of both raw and cooked tissues were then prepared in 10 volumes of 2.0 M sodium phosphate= sulphite buffer (pH 6.1). Raw and cooked tissue homogenates, soup and raw egg were extracted and assayed by HPLC for the concentrations of tetracycline agents as previously described by Gastearena et al.14 Results Antibiotic-residue positive samples were identi®ed in 23 (69.7%) out of the 33 broiler and 3 (60%) out of the 5 layer poultry farms investigated. The results of 110 raw liver samples as well as 117 raw chicken-muscle samples obtained from 22 and 23 broiler farms, respectively (liver samples from one farm were lost), indicate that one or more compounds of tetracyclines were used in these farms. Tables 1 and 2 summarize the concentrations of tetracycline compounds in raw and cooked liver respec-
tively. OXT was detectable in 90.9% of the farms and 77.3% of raw chicken-liver samples (Table 1). After cooking, the overall mean concentration of this drug increased by 45% and it was also found in a greater percentage of farms and samples (Table 2). TET, CHT and DXC were also positively detected in 46.4%, 53.6% and 33.6% of the investigated raw samples, respectively. However, unlike OXT, their concentration as well as the number of positive samples decreased after cooking. A similar pattern of results was also observed in muscle samples, with OXT having the highest concentration and being the most frequently detected (Tables 3 and 4). Table 5 summarizes the drug concentrations in the soup of cooked chicken tissue (muscle and liver). These results again show that all four investigated tetracycline agents, especially OXT, were detectable in appreciable concentrations in soup samples. Mean concentrations of OXT in raw and cooked liver were above the liver maximum residue limit (MRL) (0.30 mg=g) in the majority of residue Ð positive farms (Figure 1). In addition, the MRL for TET, CHT and DXC
301
Table 1 Summary of tetracycline drug residues in 110 raw chicken livers obtained from 22 farms
Range of farm mean conc. (mg=g) Overall mean conc. (mg=g) SD % positive farms % positive sample
OXT
TET
CHT
DXC
0.15 ± 2.93 1.00 0.78 90.9 77.3
0.15 ± 2.89 0.84 0.84 50.0 46.4
0.12 ± 3.24 0.88 0.87 59.1 53.6
0.17 ± 1.13 0.36 0.87 40.9 33.6
OXT: oxytetracycline, TET: tetracycline, CHT: chlortetracycline, DXC: doxycycline. Table 2 Summary of tetracycline drug residues in 110 cooked chicken livers obtained from 22 farms
Range of farm mean conc. (mg=g) Overall mean conc. (mg=g) SD % positive farms % positive sample
OXT
TET
CHT
DXC
0.17 ± 4.38 1.45 1.14 100 90.9
0.04 ± 0.20 0.13 0.08 18.2 12.7
0.13 ± 1.05 0.46 0.36 50.0 37.3
0.15 ± 0.20 0.18 0.02 27.3 19.1
OXT: oxytetracycline, TET: tetracycline, CHT: chlortetracycline, DXC: doxycycline. Table 3 Summary of tetracycline drug residues in 117 raw chicken muscles obtained from 23 farms
Range of farm mean conc. (mg=g) Overall mean conc. (mg=g) SD % positive farms % positive sample
OXT
TET
CHT
DXC
0.23 ± 2.05 0.96 0.54 78.3 62.4
0.11 ± 3.37 0.72 0.93 47.8 43.6
0.11 ± 2.92 0.82 0.93 43.5 38.5
0.18 ± 1.20 0.77 0.40 21.7 16.2
OXT: oxytetracycline, TET: tetracycline, CHT: chlortetracycline, DXC: doxycycline. Table 4 Summary of tetracycline drug residues in 117 cooked chicken muscles obtained from 23 farms
Range of farm mean conc. (mg=g) Overall mean conc. (mg=g) SD % positive farms % positive sample
OXT
TET
CHT
DXC
0.12 ± 2.35 1.02 0.70 73.9 58.1
0.06 ± 0.32 0.19 0.11 26.1 20.5
0.10 ± 0.93 0.43 0.30 30.4 25.6
0.20 ± 0.22 0.21 0.01 8.7 7.7
OXT: oxytetracycline, TET: tetracycline, CHT: chlortetracycline, DXC: doxycycline. Public Health
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Table 5 Summary of tetracycline drug residues in cooking ¯uid (soup) of 110 chicken muscle and liver obtained from 22 farms
Range of farm mean conc. (mg=g) Overall mean conc. (mg=g) SD % positive farms % positive sample
OXT
TET
CHT
DXC
0.08 ± 0.67 0.24 0.16 100 86.4
0.06 ± 0.30 0.15 0.09 31.8 28.2
0.04 ± 0.24 0.10 0.06 50.0 43.6
0.03 ± 0.23 0.09 0.07 31.8 23.6
OXT: oxytetracycline, TET: tetracycline, CHT: chlortetracycline, DXC: doxycycline.
Figure 1 Mean concentrations of oxytetracycline in raw and cooked chicken liver. The maximum residue limit (MRL) 0.30 mg=g.
Figure 2 Mean concentrations of oxytetracycline in raw and cooked chicken muscle. The maximum residue limit (MRL) 0.10 mg=g.
was also exceeded in raw liver samples in 9, 9 and 3 farms, respectively. However, after cooking the mean detectable concentrations of these three drugs were decreased to below MRL except for CHT in 5 farms. Mean concentrations of OXT in raw muscle exceeded the muscle MRL value (0.10 mg=g) in all 18 OXT Ð positive farms and they remained above the MRL in most farms even after cooking (Figure 2). Furthermore, mean concentrations of TET, CHT and DXC were also above the MRL in raw muscle obtained from 10, 9 and 5 farms, respectively. However, after cooking, the MRL of these drugs was exceeded only in 3, 6 and 2 of the farms, respectively. One tetracycline agent, namely TET, was also detected in 180 raw eggs collected from 3 farms. Mean levels ranged from 0.08 ± 0.25 mg=g (overall farm mean s.d. 0.15 0.04). Although 14.4% of the investigated egg samples exceeded the egg MRL value (0.20 mg=g), mean concentrations of TET in all farms were below MRL.
with the known pharmacokinetic pro®les of these drugs. Tetracyclines distribute widely into body tissues and are found in high concentrations in the excretory organs, especially the liver and in the bile. Tetracyclines undergo extensive entrohepatic circulation which leads to prolongation of their elimination half-lives; thus persisting in the body for a long time after cessation of drug administration.15 Long withholding periods for oral use of tetracyclines (up to 21 d depending on indications and type of drug) have therefore been recommended in the USA.16 OXT had the highest concentration and was the most frequently detected drug in all types of samples investigated. This may be due to the fact that long-term administration of OXT in feed is particularly recommended for controlling Mycoplasma gallispeticum infection which is considered to be one of the most common causes of chicken morbidity.15,17 However, as a feed additive the maximum recommended amount of this drug is 100 ± 200 g=ton, depending on indications, either nutritional or prophylactic.17 For economic reasons, local poultry producers add this drug to water, a practice that may enhance the absorption of the drug and ultimately its tissue concentration. The MRLs for tetracycline compounds in all foodproducing animals have been set at 0.10, 0.30 and 0.20 mg=g in muscle, liver and eggs, respectively, while
Discussion In the present study, high levels of tetracycline compounds were detected in edible chicken tissue, with liver having higher concentrations than muscle. This is in agreement Public Health
Residues of tetracycline compounds in poultry products MS Al-Ghamdi et al
ADI for humans is recommended not to exceed 3.0 mg.11,12,18,19 The results of the present study show that the overall farm mean concentrations of various tetracyclines in raw and cooked muscle were 1.9 ± 10 times greater than the recommended MRL value. Similarly, for raw liver, the overall farm means for these agents exceeded the recommended MRL by 1.2 ± 3.3 times and only the mean concentrations of TET and DXC fell below MRL following cooking. In addition, the results also show that the overwhelming majority of investigated farms (82.6% and 95.5%) had mean concentrations of at least one tetracycline compound in raw muscle and liver, respectively, in excess of MRL. Furthermore, 36.4% and 13.4% of all collected muscle and liver samples respectively had tetracycline drug levels that were above the MRL. In comparison, the percentage of residue violations for tetracyclines detected in 1777 chickens in the USA under the monitoring program mounted by the United States Department of Agriculture's Food Safety and Inspection Service (USDA=FSIS) between 1983 and 1990 was only 0.06%.16 It is currently recognized that the incidence of residue violations should not exceed 1.0% of samples and that levels above it indicate improper use of antimicrobial agents, especially the failure to adhere to recommended withdrawal times.16 The effect of cooking on antibiotic residues was also investigated in the same samples. The results showed that the overall mean levels of TET, CHT and DXC in both muscle and liver decreased markedly after cooking. On the other hand, the overall mean levels of OXT in cooked tissues were higher than in raw ones but the difference does not reach statistical signi®cance (P > 0.05). This may re¯ect the high stability of OXT to heating. It has been documented that OXT crystals remain potent after heating for 4 d at 100 C.20 However, the overall farm mean levels of all four agents in cooked muscle remained above the internationally recommended MRL while only those of TET and DXC fell below MRL in cooked liver. Extensive use of multiple tetracycline agents in broiler farms was also detected in this study with 73.9% and 95.5% of investigated farms testing positive for 2 or more tetracyclines in raw muscle and liver, respectively. While OXT and CHT have been approved in the USA for nutritional, prophylactic and therapeutic uses in poultry, TET and DXC are to be used only as therapeutic agents.17 In the present study, all four tetracyclines were detectable at the time of slaughter, indicating that the four agents were being continuously used throughout the production cycle. Multi-tetracycline use is not clinically justi®able as these drugs share the same mechanism of action and cover the same microbial spectrum. This practice may contribute signi®cantly to the development of microbial resistance to the drugs. Indeed, our own results (unpublished data) showed that 89.2% of 1962 chicken normal ¯ora isolates were resistant to tetracyclines, while on the other hand 52.8% of human pathogenic organisms, investigated for tetracyclines susceptibility in a referral hospital in the region, were resistant to this drug. Many reports indicate that microbial resistance to antibiotics may arise as a result of animal exposure to antimicrobial agents with the resistance being possibly transferred to human pathogens.5,7,8,21 ± 23 In addition, human exposure to animal products containing signi®cant levels of antibiotic residues may provoke immunological responses in susceptible individuals9 and cause disorders of intestinal ¯ora.8,10
Only one tetracycline agent (TET) was detected in eggs and with mean levels in all 3 positive farms below the recommended MRL. However, this may be due to growers' awareness of the impact of tetracycline administration on calcium absorption which is essential for egg production. In conclusion, the results of this study indicate that there is widespread misuse of tetracycline agents by poultry producers in the eastern province of Saudi Arabia, possibly re¯ecting a general lack of compliance with internationally recommended practices regarding the use of antimicrobial agents. This malpractice includes multiple use of drugs belonging to the same pharmacological group and lack of implementation of recommended withdrawal times. It stresses the need for stricter regulations for the use of antimicrobial drugs in the poultry industry as well as inspection of chicken for drug residues prior to marketing.
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Acknowledgements This work was funded by the King Abdulaziz City of Science and Technology (KACST), Saudi Arabia (Grant no. AT-15-79) to whom we express our gratitude. Our sincere thanks also go to Dr Abdul-Rahman Qurashi, and the technical staff at the Microbiology laboratories at King Fahd Hospital of the University and College of Medicine for their assistance.
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