Antibiotic Residues of Penicillin and Oxytetracycline in Yoghurt: One Health Needs Paradigm Shift, Legislation and Policy Formulation in Pakistan

According to Food and Drug administration in the United State, Residues are substances and their metabolites which are present in staples acquired from animals to which the drugs being referred to have been directed (Lecuona et al., 2022). With the discovery of pharmaceuticals, pharmaceuticals have been being used tremendously that include antibiotics (ABs). The discovery of animal and environmental health requires the exigent attention from all the states around the world (Fatoki et al., 2018) Antibiotic agents can kill or restrain the development of defenseless bacteria, permitting resistant isolates to colonize and duplicate in a provided climate (Saraiva et al., 2022).

One Health (OH), is closely connected with the health of animal, human beings, and the environmental health (Acharya et al., 2020). The worldwide emerging OH approach is an essential to address this issue effectively. The study has revealed that the issues which are related to the accumulation of antibiotics residues from different sources is becoming part of waste-water are eventually leading to the antibiotic resistance (AR) (Sorinolu et al., 2021). Retailers and merchants do not have satisfactory information on compelling dose and the conceivable results of veterinary medications (Samad, 2022). Abuse of these antibiotics in animals has prompted the presence of residues in determined food sources, like milk and dairy items (Lecuona et al., 2022).

Tetracyclines are wide range and the most normally involved antibiotic agents for prophylactic and development purposes in numerous nations all over the planet, with oxytetracycline (OTC) being one model (Kabrite et al., 2019). The first antibiotic to be utilized in veterinary medication was penicillin G, which was at first made accessible in 1947 (Samad, 2022). Misuse of this antibiotics may led to the resistance and residues in dairy products (Pourtaghi et al., 2022). Poor quality milk containing the ABs poses threat to the one health and public health. The quality of milk from safety perspective should be improved (Nyokabi et al., 2021).

In contemporary world, the Antibiotic Resistance (ABR) is one of the serious international threat to the human health inflicting many thousands fatalities each year due to the antibiotic residues in a variety of products (Zhou et al., 2018). Different insightful techniques have been depicted to decide antibiotics residues in milk, for example, microbiological, chromatographic, immunochemical, receptor also, enzymes based tests(Kaya and Filazi, 2010). Milk is an exceptionally polished off food on the planet which has likewise an extraordinary incentive for human wellbeing Buildups of anti-toxins are predominantly tracked down in milk because of their imprudent use in treating irresistible illnesses of animals (Sachi et al., 2019).

All vital regulatory, economic, and political forms acknowledge antibiotic resistance (ABR) as one of the crucial universal health challenges of the twenty-first century (Hernando-Amado et al., 2019). According to a recent study, over 1 million deaths are caused by antibiotic resistance (Jamrozik and Heriot, 2022). The health warning of ABR is especially concerning in low and middle income countries due to the increased probability of local area obtained safe contaminations, the more infectious disease burden in the common population, lack of coverage of pure water and sanitation, and implementation of antibiotics in foodstuff production and healthcare (Rousham et al., 2018). According to data collected, the antibiotic resistance crisis has worsened since the twenty-first century, and there is a need to reduce the burden of antibiotics in order to avoid antibiotic resistance (Klein et al., 2018). Otherwise, it will have a negative impact on the OH (van-Puyvelde et al., 2018). Policy makers should think about the OH to provide candid benefits. The scientists can inculcate a variety of sectors to work in collaboration for the OH (Balkhy et al., 2018). The implementation is another challenge and it should be in letter and spirit regarding the OH approach. China has initiated in a strict manner to conduct legal clinical practices since then to improve the relationship among the triad of planet (Fang and Song, 2021).

The key objectives of the study were to detect commonly used antibiotics like penicillin and oxytetracycline as a one health issue and suggesting a way-forward for the legislatives to ensure food safety through food controlling authorities.

The yoghurt samples were collected from a variety of areas of Lahore, District of Punjab Province, Pakistan. These areas included as sub-areas of Lahore district as Baad-Shahi Mosque to Sundar Estate Area and Johar Town to DHA.

The experimental source included 90 samples of yoghurt as fifty grams per sample (50g/sample). The samples were collected from the open markets of Lahore, and brought to UVAS, Lahore.

The HPLC for yoghurt samples can be easily used as a one of the most reliable and modern technique. The HPLC MS/MS has become gold- standard for multi residue analyses because of the fact that it has vintages over other methods. It is considered one of the most sensitive and reliable method for testing the samples in context of ABs detection and quantification. One of the best sensitive techniques includes HPLC-MS/MS (Lu et al., 2019). The HPLC and LC-MS/MS are highly sensitive detection methods for ABs in food (Majdinasab et al., 2020). A variety of accurate analytical methods are available for the purpose of detection of the ABs. These techniques include high performance liquid chromatography (HPLC), gas chromatography, GC-MS and rapid chromatographic detection kits (Teixeira et al., 2020). The samples of yoghurt were collected in plastic zipper bags. Those were transported to the university’s clinical medicine laboratory in ice boxes. For preservation purpose, the samples were kept in refrigerator prior to testing. The quantification of the ABs residues was performed which under HPLC and was assessed statistically through a variety of available software. The processing of samples was initiated and completed as per standard procedures provided with HPLC machine and equipment (Figure 1). The quantification of the ABs residues was performed which under HPLC and was assessed statistically through a variety of available software.

SPSS (IBM Corp., NY, USA) version 26.0 was used to encode the data set. Reports of baseline characteristic frequencies were made. All variables were originally investigated using a univariable analysis to identify the risk factors linked to the incidence of antibiotic residues in samples. The correlation between antibiotic residue and the positive samples was evaluated using Chi-square (χ2). Multivariable logistic regression (MLR) was also employed to identify factor linked with positive and negative dichotomous sample outcome. The 95% CI was used to emphasize the odds ratio in the MLR-based statistical test, which was based on the two-sided Wald test.

The 23 samples out of 90 were found positive making a percentage of 25.56 and 67 were negative making a percentage of 74.44 for Penicillin and Oxytetracycline (ABs) and both of ABs simultaneously (Table 1).

Table 1: Yogurt positive and negative samples with percentages.

The yogurt contained as high as 13.95 µg/Kg or ng/g penicillin as the highest quantity in any of the samples while the lowest penicillin was detected as 7.07 µg/Kg or ng/g in a sample. Similarly, the average highest penicillin was figured out in a sample as 10.53 µg/Kg or ng/g. On the other hand, in case of oxytetracycline, the highest quantity was observed as 306.73 µg/Kg or ng/g in a sample while the lowest quantity of oxytetracycline was figured out in a sample as 197.77 µg/Kg or ng/g. On similar grounds, the average highest oxytetracycline was observed as 255.88 µg/Kg or ng/g in the samples while the highest quantities of both of the Abs simultaneously (penicillin and oxytetracycline) were figured out as 12.63 simultaneously µg/Kg or ng/g penicillin and 275.41 µg/L or ng/ml as oxytetracycline , lowest both penicillin oxytetracycline were figured out as 9.90 µg/Kg or ng/g penicillin and 258.72 µg/Kg or ng/g as oxytetracycline respectively, average for both penicillin and oxytetracycline, penicillin as 10.90 µg/Kg or ng/g and 248.73 µg/Kg as oxytetracycline as well (Table 2 and Figure 2).

Table 2: Delineating penicillin and oxytetracycline in yogurt samples µg/kg.

The said samples are being presented as mean ± standard error of mean described by Coles. The obtained data was analyzed by using statistical technique known as ANOVA through the software named as SPSS, version 20.20. The mean values were sought as 10.606, and 254.09 while the standard deviation values were calculated as 1.907 and 33.59. The p-value obtained is 0.000000001 for yogurt only for the positive samples which have been quantified. This data has depicted that significant difference patterns have been shown among all of the positive samples. The positive samples had different quantities were present in different samples (Table 3).

Table 3: ANOVA of yogurt positive samples.

The 23 samples out of 90 were figured out to be positive making Penicillin at the highest 11 out of 23, making a percentage of 47.83 %, Oxytetracycline 9 out of 23, making a percentage of 39.13% and both Penicillin and Oxytetracycline simultaneously, making a percentage of 13.04% (Table 4 and Figures 3, 4, 5, 6, 7).

Table 4: Yogurt positive samples- a drug wise comparison.

As per following objectives of the current studies have been achieved which could be extended prospectively by discovering new avenues through multidisciplinary vistas:

• Grave kind of effects of antibiotic residues were figured out from the analyzed data which were present in yoghurt on national food safety and public health eventually lead to one-health issue. This is due to the abuse of ABs in livestock which remains unmeasured at the hands of quackery.
• The pragmatic way-forward has been suggested for all kind of stakeholders. These stakeholders include legislative, judiciary, corporate sector, common public (consumers) and prescription policy makers.
• Public awareness-based newspapers article has been published in national dailies, on national TV channels and social media channels.
• There is a strict need of policy in both private and public sector to reduce the use of ABs in live-stocks of Pakistan.

Antibiotics (ABs) are commonly exploited to treat sickness in animals and to enhance their production. These substances can be found as residues in milk, and such kind of food is unsuitable for human consumption. The tenacious and strict control of those Abs through legislation would be indispensable and it must be checked with iron hands (Ngangom et al., 2019). The utilization of ABs in cattle for the treatment has contributed to the residues in dairy items. Penicillin is ordinarily utilized veterinary medication to treat mastitis in dairy cows (Worku et al., 2017). A range of sensitive and specific strategies are developed for the quantification of those compounds in numerous livestock food materials. However, it is pertinent to mention here that the determination of trace residues in foods like milk typically need intensive sample extraction and preparation before conducting instrumental analysis (Pérez-Valdespino et al., 2020). Twenty three (23) out of 90 samples were positive for antibiotic (Penicillin and Oxytetracycline) residues according to techniques applied as mentioned in Table 1 and same technique was used by (Abebew et al., 2014). Antibiotic residues are present in different samples of milk with different percentages as mentioned by (Ambaw and Aytenfsu, 2021) and (Alnassrallah et al., 2022). In proportion of 23 positive samples, 11 out of 23 were positive for penicillin residues making a percentage of 47.83% and 9 out of 23 were positive for oxytetracycline making a percentage of 39.13% and for both were 3 out of 23 making a percentage of 13.04% as mentioned in Table 2. The quantification of the ABs residues was performed which under HPLC and was assessed statistically through a variety of available software (Figure 1). These results are different from (Kabrite et al., 2019) having penicillin percentage is 0 % and oxytetracycline is of 50%. The reason could be the difference in samples size, sample area, methodology and techniques applied and the environment of that area and least access of antibiotics to livestock in that area.

The data regarding the presence of commonly used ABs i.e. penicillin and oxytetracycline in livestock which are used as routine food for human consumption has delineated presence of antibiotic residues in yoghurt samples collected from the open markets of Lahore. There is a dire need of policy formulation in both private and public sector to reduce the use of ABs in live-stocks of Pakistan. Furthermore, all the stake-holders may work in collaboration to ensure OH.

Profound gratitude for the team of ABN TV’s Morning Show (Mr. Muneeb Hamid, Ms. Nimra Abbas and Ms. Farwa Raza) and digital platform (Mr. Tabasum Ranjha) for creating public awareness and disseminating the current research endeavours among the stake holders.

The authors have declared no conflict of interest.