Transmission of Administered Amoxicillin Drug Residues from Laying Chicken to their Commercial Eggs

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1 Transmission of Administered Amoxicillin Drug Residues from Laying Chicken to their Commercial Eggs Walaa O. Khattab, Hesham B. Elderea, Eglal G. Salem, Naglaa F. Gomaa Food Hygiene and Control, Nutrition department, High Institute of Public Health, Alexandria, Egypt ABSTRACT Background: Antimicrobial agents in food producing animals are a very important public health issue. They are routinely added to animal feed at sub-therapeutic levels for their growth promoting properties. This practice, however, carries many disadvantages, such as the stimulation of microbial resistance to antibiotics. Aim: This study was carried out to determine administered Amoxicillin residues in laying chickens and in their commercial eggs, and the effect of cooking and storage condition on Amoxicillin residues in their eggs. Material and Methods: A total of 50 terminal faeces were collected from laying hens in addition, a total of 215 commercial egg samples were randomly selected from the farm treated with Amoxicillin. Microbiological agar diffusion method was used. Results: the withdrawal time of Amoxicillin was 7 days. Its residues were detected in both egg yolks and egg whites for 6 successive days after the last drug administration. In addition, Amoxicillin residues remained until the 7th day from drug administration in eggs stored at room temperature, and at 4 C. Amoxicillin residue was not affected after boiling eggs for 10 minutes. Conclusions and Recommendations: eggs should not be distributed to markets until the end of the drug's withdrawal period. It is also recommended to observe the withdrawal period of drugs before poultry slaughter or table egg distribution to avoid antimicrobial resistance and to inform both owners and consumers about the risks of antibiotic residues in egg contents. Keywords: Antimicrobial agents, amoxicillin residues, laying chicken, poultry eggs, withdrawal period. Corresponding author: Dr. Naglaa F. Gomaa Food Hygiene and Control, Nutrition Department

2 High Institute of Public Health Alexandria university, Egypt INTRODUCTION There can be no doubt that antibiotics have proved of immeasurable benefit to both man and domesticated animals. Their principal uses are the prevention and therapy in man and his animals raised under intensive husbandry methods of production. (1, 2) In addition, they are routinely added to animal feed at sub-therapeutic levels for their growth promoting properties. (3) Thus, these agents are considered very valuable in preventing major economic losses to the grower caused by disease outbreaks. (4) This practice, however, carries many disadvantages, such as the stimulation of microbial resistance to antibiotics, with the possible transfer of resistant pathogens from animal and animal products to human, (5) which may pose a major health risk to the public. (6) Veterinary drugs are therapeutically used for laying hens, generally by mass application via water or feed or may reach them accidentally, for example, as a result of cross contamination in the feed mill. When these compounds reach the blood stream, they are distributed over the whole body, especially the ovary with growing follicles and the oviduct, where the egg white is formed and secreted, (7,8) and therefore may increase the incidence of unacceptable residues in eggs. Such residues are reduced by establishing and adhering to withdrawal periods before slaughter, and by sometimes prohibiting the use of certain antibiotics in laying hens. (9) 298

3 This study aimed to determine the administered Amoxicillin residues in laying chickens, and their commercial eggs, and to study the effect of cooking and storage condition on the amount of Amoxicillin residues in eggs. MATERIALS AND METHODS MATERIALS The laying hens in one egg producing farm -Alexandria, Egyptwere medicated with Amoxicillin. The antibiotic was added to hen's drinking water (25mg / hen for 3days). METHODS 1. Sampling 1.1 Samples of laying chicken faeces A total of 50 laying hens treated with Amoxicillin were randomly selected from the farm. Terminal faeces were collected from each laying hen into a sterile 10ml glass test tube and transported on ice to the laboratory for antimicrobial drug residue determination. 1.2 Samples of commercial eggs A total of 215 commercial egg samples were randomly selected from the previously mentioned Amoxicillin treated farm for Amoxicillin residues determination. These were divided as follows: A) A total of 145 commercial egg samples were collected during the period of drug administration (2 days from the beginning of medication) as follows: * 50 commercial eggs were taken to the laboratory for immediate examination or refrigerated at 4 o C till the next day. 299

4 * 40 egg samples stored for one month at room temperature * 40 egg samples stored in the refrigerator (4 o C) for one month. All stored eggs were examined as 10 eggs at the end of the 7 th, 14 th, 21 st and 28 th days of storage. * 15 commercial eggs. These were boiled for 10 minutes then examined for the presence or absence of Amoxicillin residues. B) A total of 70 commercial eggs randomly selected from the farm after the end of the period of drug administration. These were collected as 10 egg/day for 7 successive days and transported to the laboratory for immediate examination. 2. Samples preparation * Chicken faeces samples Chicken faeces were first purified to about 1 gm of faeces; 5 ml of sterile phosphate buffer (ph 7.0) was added and mixed thoroughly by shaking. The suspension was then placed in a boiling water bath for 15 minutes to reduce bacterial interference during the microbial inhibition test. After heating, the sample suspension was centrifuged at 3000 rounds per minute for 10 minutes. The supernatant fluid was used to inoculate sterile filter paper discs (12mm) for the test. * Raw egg samples Eggs were washed with a brush using soap and warm water, then drained and immersed in 70% alcohol for 10 minutes. Then they were removed from alcohol, drained and flamed. Using sterile gloves, egg meat was aseptically removed by cracking the egg with a sterile breaking knife. Separation of egg white and egg yolk was done using a sterile commercial separator or a sterile spoon. The yolk was separated from excess egg white by rolling it on a sterile towel. The albumin and yolk were then drained out carefully and transferred into separate 300

5 sterile transparent polythene bags. Ten milliliters of phosphate buffer (ph 7.0) were added to each of the albumin/yolk bag and homogenized or blended together using a stomacher lab-blender 400. (10,11) * Boiled egg samples Each egg was washed with a brush using soap and water warmer than the egg. The egg samples were boiled for 10 minutes in a water bath. The boiled eggs were handled with sterile gloves to aseptically remove egg meat by cracking the egg with a sterile breaking knife. Separation of egg white and egg yolk was done using a sterile spoon. The albumin/yolk was then transferred into a sterile transparent polythene bag. Ten milliliters of phosphate buffer (ph 7.0) were added and homogenized or blended together using a stomacher lab-blender Testing Screening the antimicrobial drug residues were conducted by using microbiological agar diffusion method with a suitable test organism. (12,13) Bacillus cereus, Escherichia coli, and Staphylococcus aureus were used as test organisms to test faeces and commercial eggs for Amoxicillin residues. These were isolated and identified in the laboratory of the microbiology department of the High Institute of Public Health. Pure inocula were obtained by selecting four to five colonies of the same morphology, inoculating them to a broth medium, and allow the culture to achieve good active growth by incubation at 37 C for 18hrs. Broth suspension of each of the test organism was adjusted with a sterile physiological saline solution to a concentration approximately equal to the turbidity of Barium Chloride McFarland standard No.1 equivalent 301

6 to cells/ml. (14) Amoxicillin (10 mg) susceptibility test discs (OXOID) were used as control discs. Mueller-Hinton agar (MHA) (DIAGSERA) plates were inoculated with the test organism by dipping sterile cotton swab sticks into each of the 18hrs broth suspensions of Bacillus cereus, Escherichia coli, and Staphylococcus aureus until it was saturated with the suspension. The plates were then gently and thoroughly swabbed uniformly, to achieve a confluent growth. Inoculated plates were allowed to dry for about 5-10 minutes. With a sterile thump forceps, the discs impregnated in the chicken faeces samples were gently placed on the surface of the MHA. Similarly, using a sterile forceps, paper discs were dipped into the albumin and yolk separately and allowed to soak, excess albumin and yolk was drained from the disc. Samples and Amoxicillin control discs were firmly but gently pressed on the surface to allow for proper diffusion on the agar around the discs. After that, plates were incubated at 37 C for hrs and then viewed for the presence or absence of zones of inhibition of test organism around the test and control discs. Any disc with a zone of inhibition of, or greater than 1mm around the disc was considered positive for the presence of antimicrobial drug residues. RESULTS Table (1) demonstrates that 48 (96%) out of the 50 collected faeces samples were positive for antibiotic drug residues against each of the test organisms. A small percentage (4%) of the plates was negative for Amoxicillin residues when tested against each of Bacillus cereus, Escherichia coli and Staphylococcus aureus. 302

7 Table (2) shows that the percentage of egg white samples positive for Amoxicillin residues was higher than that of egg yolk ones. This was shown in all of the test bacteria as 56, 62 and 60 egg white samples, whereas 46, 52 and 50 egg yolk samples were positive for Amoxicillin residues when tested against Bacillus cereus, Escherichia coli and Staphylococcus aureus, respectively. It can be also seen from the table that all of the 15 boiled egg yolk and egg white samples were positive for Amoxicillin residues when tested against the same bacterial species. Table (3) shows that Amoxicillin residues were sometimes not detected in egg yolk samples but detected in egg white and vice versa. This is shown in the eggs collected at the second day of the withdrawal period, where Escherichia coli growth was inhibited in 6 (60%) of the tested egg white samples and in the same time was inhibited in 5 (50%) of the tested egg yolk of the same 10 collected eggs. Bacillus cereus, on the other hand was inhibited in higher number of egg yolk (40%) than in white samples (20%) during the third day of withdrawal. These percentages showed some fluctuations as they were raised to 80% and 60%, respectively during the fourth day of withdrawal, decreased during the fifth day (0% and 20%, respectively), then re-increased during the sixth day (40% and 20%). These fluctuations were also observed for the other test organisms. Moreover, egg yolks during the 5 th day of the withdrawal period were negative for Amoxicillin residues while 2(20%) of egg whites were positive when tested against Bacillus cereus. On the 6 th day of withdrawal, Escherichia coli was found to be inhibited in 5 (50%) egg yolk samples but all egg whites were negative. At the seventh day, there were no detectable Amoxicillin drug residues. 303

8 Table (1): Bacterial Inhibition by Chicken Faeces Samples Collected 2days After Amoxicillin Drug Administration. Bacteria Bacterial inhibition by Amoxicillin residue Positive plates Negative plates No. % No. % Bacillus cereus Escherichia coli Staphylococcus aureus n= 50 chicken faeces samples. Table (2): Bacterial Inhibition by Egg Samples Collected 2 days After Amoxicillin Drug Administration. Egg samples Bacteria Bacterial inhibition by raw eggs (n=50) Positive plates Negative plates Bacterial inhibition by boiled eggs (n=15) Positive plates No. % No. % No. % Egg yolk Egg white Bacillus cereus Escherichia coli Staphylococcus aureus Bacillus cereus Escherichia coli Staphylococcus aureus

9 Table (3): Day 1 st day 2 nd day 3 rd day 4 th day 5 th day 6 th day 7 th day Bacterial InhibitionCcaused by Egg Samples ( Yolk and White) in Egg Samples Collected During the Withdrawal Period of Amoxicillin. Bacteria used Yolk (n=10) Egg content White (n=10) Positive bacterial inhibition by Amoxicillin residue No. % No. % Bacillus cereus Escherichia coli Staphylococcus aureus Bacillus cereus Escherichia coli Staphylococcus aureus Bacillus cereus Escherichia coli Staphylococcus aureus Bacillus cereus Escherichia coli Staphylococcus aureus Bacillus cereus Escherichia coli Staphylococcus aureus Bacillus cereus Escherichia coli Staphylococcus aureus Bacillus cereus Escherichia coli Staphylococcus aureus Table (4) indicates that the number of positive Amoxicillin residues in egg yolk and egg white samples were similar in eggs samples stored for 7 days at room temperature and those stored for 7 days at 4 C, when 305

10 14, 21, and 28 Days 7 Days Storage Period J Egypt Public Health Assoc Vol. 85 N o. 5 & 6,2010 the eggs were tested against Bacillus cereus, Escherichia coli and against Staphylococcus aureus. Escherichia coli was the most inhibited (80% of egg yolk and 90% of egg whites samples), followed by both Bacillus cereus and Staphylococcus aureus (70% of egg yolks and 60% of egg whites, respectively). After 14, 21, 28 days of storage, no Amoxicillin residues were detected. Table (4): Effects of Storage of Egg Samples on Amoxicillin Residues After 7, 14, 21, and 28 days. Egg content Yolk White Yolk White Bacteria Storage at room temperature n=10 Storage at refrigerator (4 o C) n=10 Bacterial inhibition by Amoxicillin residue No. % No. % Bacillus cereus Escherichia coli Staphylococcus aureus Bacillus cereus Escherichia coli Staphylococcus aureus Bacillus cereus Escherichia coli Staphylococcus aureus Bacillus cereus Escherichia coli Staphylococcus aureus Table (5) shows that Amoxicillin is secreted at almost similar concentrations in chicken faeces (mean inhibition zones ranging from 1-1.2cm for the 3 test organisms) and in raw egg yolks and whites (mean inhibition zones ranging from cm). Eggs boiled for 10 minutes, on the other hand, had higher mean inhibition zones than those induced 306

11 by raw eggs (mean inhibition zones from 1.4-2cm for boiled egg yolks and whites). The minimum mean inhibition zones were seen in plates inoculated with Bacillus cereus (from cm for raw and boiled eggs, respectively). Following were those in plates inoculated with Staphylococcus aureus (from 1-1.5cm), then were those of Esherichia coli (from cm). Table (5): Minimum, Maximum and Mean Inhibition Zones Induced by Chicken Faeces and Egg Samples Collected from the Farm Treated with Amoxicillin. Sample Bacteria used Inhibition zone (cm) Minimum Maximum Mean Chicken faeces n=10 Raw egg yolk n=10 Raw egg white n=10 Boiled egg yolk n=10 Boiled egg white n=10 Bacillus cereus Escherichia coli Staphylococcus aureus Bacillus cereus Escherichia coli Staphylococcus aureus Bacillus cereus Escherichia coli Staphylococcus aureus Bacillus cereus Escherichia coli Staphylococcus aureus Bacillus cereus Escherichia coli Staphylococcus aureus

12 DISCUSSION Amoxicillin is one of the aminopenicillins which are semi synthetic derivatives of Beta-Lactam antibiotics that have free amino group at the α-position at R on the penicillin nucleus. They exert their bactericidal effects by preventing bacterial cell synthesis and disrupting bacterial cell wall integrity. (14) Antimicrobial residues in chicken faeces samples Amoxicillin residues were detected in 96% of chicken faeces samples when added at 25mg/kgBW in drinking water. The three tested bacteria were sensitive to amoxicillin and induced inhibition zones that ranged from cm for Bacillus cereus, cm for Escherichia coli and for Staphylococcus aureus (Table 5). This result is in agreement with the fact that aminopenicillins and their metabolites are excreted in the urine. (15) The urinary system of the chicken does not contain a urinary bladder. Ureters carry the urinary waste to the cloacae which is excreted along with the faeces. (16) Thus, Amoxicillin residues were found in chicken faeces. For most veterinary drugs, the presence of drugs in blood, faeces, or urine at the time of slaughter indicates presence of drugs in edible tissues albeit at lower concentrations. Hens treated with drugs would produce eggs containing residues for some time even after drug withdrawal. (17) This is of concern because backyard poultry rearing does provide a continuous supply of eggs to many households. Meanwhile there is no official residue monitoring program and consumer response towards the dangers posed by residues is passive. (18) Thus, there is a risk of sustained consumer exposure to residues. 308

13 Antimicrobial residues in commercial egg samples collected from the farm treated with Amoxicillin during the drug administration (2 days after treatment) In this study, Amoxicillin residues were detected in both egg yolk and egg white during drug administration when Amoxicillin was administrated at 25mg/kgBW in chicken's drinking water for three successive days. After intestinal absorption, transport via blood plasma is responsible for deposition of drugs in yolk in the ovary or egg white in the oviduct. Physicochemical properties of the drugs, the physiology of the hen and egg formation will determine how much drug will be deposited. (8) For egg yolk samples, 46% of the examined samples were positive when tested against Bacillus cereus with zones of inhibition ranging from cm, 52% were positive when tested against Escherichia coli with zones of inhibition ranging from cm, while 50% of them were positive when tested against Staphylococcus aureus with zones of inhibition ranging from cm (Tables 2 and 5). These results agreed with another study where it was reported that a single exposure to a drug might be sufficient to detect the drug in either egg white or yolk depending on the characteristics of the drug and the sensitivity of the analytical method used. (8) Regarding egg white samples, the percentages of positive samples were higher. These were 56% against Bacillus cereus with zones of inhibition ranging from cm, 62% were positive when tested against Escherichia coli with zones of inhibition ranging from cm, while 60% of them were positive when tested against Staphylococcus aureus with zones of inhibition ranging from cm (Tables 2 and 5). These results agreed with the fact that the residues of drugs occur first in egg white. Residues in egg white are a reflection of plasma levels and 309

14 will therefore show a constant level over time when plasma levels do. The time needed to achieve a constant level in egg white is generally 2-3 days. (8) This variation in the positive percentages between the three test bacteria may be due to their varying degree of resistance to Amoxicillin. Aminopenicillins are known to be active against non-penicillinase producing Staphylococci species, against some gram positive bacilli and they are also active against Enterobacteriaceae, including strains of Escherichia coli. (19) Results also showed that the raw egg yolk inhibition zones of most of the test bacteria were slightly higher than those of egg white ones (Table 5). Others found much higher residues of ampicillin (which is also an aminopenicillin but has a lower resistance to gastric acid and lower absorbance than Amoxicillin) in egg yolk (0.025 mg/kg) than in egg white (0.008 mg/kg) when laying hens were exposed to 1.5g/litter in drinking water for 5 successive days. (20) Antimicrobial drug residues in commercial eggs collected from the farm treated with Amoxicillin during the withdrawal period (2 days after treatment) In this part of the study, amoxicillin drug residues were tested in both egg yolk and egg white for 7 successive days after drug administration to detect the withdrawal time. Although the half-life of all aminopenicillins is approximately minutes and it has an extremely short plasma half-life in laying hens, (21) Amoxicillin residues remained in eggs for 6 days after drug administration, however, at the seventh day, there were no detectable amoxicillin drug residues (Table 3). Disappearance of drugs from white and yolk depends heavily on the plasma levels of the drug tested. Drugs that clear rapidly from the 310

15 body also disappear from egg white in 2-3 days after cessation of exposure. Disappearance of drugs from yolk generally takes 10 days. (8) Amoxicillin residues were found also in egg white for 6 successive days after drug administration but generally at lower percentages than egg yolk beginning from the third day of withdrawal (Table 3). The nature of the tissue can play an important role in drug disposition. In case of the laying hens, the dynamic process of yolk formation significantly influences incorporation and storage of drug residues. Individual egg yolks develop over an extended period with the latter period of rapid yolk accumulation occurring approximately 2 wk before ovulation. Additionally, there are a number of smaller yolks waiting to enter the rapid growth phase that are approximately 2 to 6 wk from ovulation. (22-24) It is probable that these residues were incorporated and stored during the dosing period in pre ovulatory yolks for a number of days before ovulation and formation into a whole egg. Similarly, hens injected for 1, 2 and 3 days with ampicillin were found to produce eggs containing detectable ampicillin residues for 6 days after the last injection. The study demonstrated that drug residues were contained in eggs laid a number of days after drug withdrawal and because plasma ampicillin was not detectable even 24 h after final dosing, it was concluded that the majority, if not all, of the incurred ampicillin residues contained in eggs laid after drug withdrawal were due to incorporation and storage of drug in pre ovulatory yolks during the dosing period. (25) Effect of storage of eggs at room temperature as well as in the refrigerator at 4 C on Amoxicillin residues In this section eggs were collected from the farm at the last day of drug administration one half was stored at room temperature and the 311

16 other one was refrigerated at 4 C for a period of one month. Every week eggs were tested for Amoxicillin residues in both egg yolk and egg white. Results showed that, at the 7 th day of storage, eggs stored at room temperature and those stored refrigerated at 4 C, were positive for amoxicillin drug residues in 70% of egg yolks when tested against Bacillus cereus and Staphylococcus aureus and in 80% of the egg yolks when tested against Escherichia coli. For egg white, amoxicillin drug residues were detected in 60% of egg whites for Bacillus cereus and Staphylococcus aureus and in 90% of the egg whites for Escherichia coli. Amoxicillin drug residues were not detected at the 14 th, 21 st, 28 th days of storage (Table 4). Amoxicillin drug has a short half-life and poor tissue stability. (26) This explains the detection of Amoxicillin residues in laid eggs only at the 7 th day of storage, in addition to their disappearance from egg yolks and egg whites while storage at room temperature and at 4 C. Effect of eggs boiling for 10 minutes on Amoxicillin residues. Amoxicillin drug residues were detected in all of the egg yolks and egg whites after egg boiling for 10 minutes when tested against Bacillus cereus, Escherichia coli and Staphylococcus aureus (Table 5). In another study, heating for min at 100 C was required to inactivate tetracycline in eggs. This is longer than the min required to hard boil eggs. (27) Other drugs including Streptomycin and Neomycin in eggs were similarly found to be stable to ordinary cooking procedures such as frying, poaching, scrambling and hard boiling. (28) Others found that Lasalocid was less stable when heated in eggs rather than in muscle, presumably for the higher ph of eggs. (29) 312

17 It is worth mentioning that the mean inhibition zones obtained from boiled egg yolks and whites (1.4-2cm) were higher than those of raw egg yolk and whites (from cm) as indicated in table 5. Thus egg boiling for 10 minutes did not influence Amoxicillin residues. Similarly, cooking for 10 minutes did not significantly influence Naracin residues in eggs. (31) This may reflect the high stability of Amoxicillin to heating. Others found that the overall mean levels of tetracycline, Chlortetracycline and Doxycycline in both muscle and liver were found to decrease markedly after cooking. On the other hand, the overall mean levels of Oxytetracycline in cooked tissues were found to be higher than raw ones reflecting the high stability of Oxytetracycline to heating. (4) CONCLUSION AND RECOMMENDATIONS It can be concluded that: The withdrawal time of Amoxicillin is 7 days. Its residues were detected in both egg yolks and egg whites for 6 successive days after the period of drug administration. Amoxicillin residues remained until the 7 th day from drug administration in eggs stored at room temperature, at 4 C. Amoxicillin residues were not affected by boiling eggs for 10 minutes. Escherichia coli was more sensitive to Amoxicillin than the other test bacteria. 313

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