Isolation of ESBL E. coli From Slaughtered Chicken of Open Market in District Abbottabad

Mehwish Malik1*, Zanib Sadia2, Muhammad Sajid3, Hammidullah1, Yasir Amin1, Zubair Ali1 and Sohail Ahmad1

1Veterinary Research and Disease Investigation Center Mandian, Abbottabad, Pakistan; 2University of, Haripur, Haripur, Khyber Pakhtunkhwa 22620, Pakistan; 3Poultry Research Institute, Jaba, Mansehra, Pakistan.

Editor | Muhammad Abubakar, National Veterinary Laboratories, Park Road, Islamabad, Pakistan.

Received | September 14, 2022; Accepted | May 29, 2023; Published | June 16, 2023

*Correspondence | Mehwish Malik, Veterinary Research and Disease Investigation Center Mandian, Abbottabad, Pakistan; Email: dr.mehwish2015@gmail.com

Citation | Malik, M., Z. Sadia, M. Sajid, Hammidullah, Y. Amin, Z. Ali, and S. Ahmad. 2023. Isolation of ESBL E. coli from slaughtered chicken of open market in district Abbottabad.. Veterinary Sciences: Research and Reviews, 9(1): 82-86.

DOI | https://dx.doi.org/10.17582/journal.vsrr/2023/9.1.82.86

Keywords | Poultry, ESBL, E. coli, Antibiotics

Copyright: 2023 by the authors. Licensee ResearchersLinks Ltd, England, UK.

This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

Introduction

Antibiotic are the agent which are majorly used for bacterial and parasitic infections. Penicillins, Cephalosporins, Tetracyclines, Aminoglycosides, Macrolides, Quinolones, Sulfonamides and trimethoprim are common group of antibiotics used worldwide. Escherichi coli (E. coli) is bacteria that exist commensally in the intestine of animals and humans, but pathogenic strains cause disease in poultry (Sarba et al., 2019). It is major cause of Colibacillosis, which is a disease of economic importance in poultry through decreasing productivity, carcass quality, increase mortality, and prophylaxis and treatment cost and is reported worldwide (Lutful, 2010). ESBL E. coli is a gram negative microbe belongs to family enterobacteracace comprising of other bacteria i.e., Shigella, Proteus, Serratia, Klebsiella, Enterobacter, Citrobacter, Salmonella and other species belongs to this family (Jaspal et al., 2013; Teklu et al., 2019; Coudron et al., 1997). Extended-spectrum beta-lactamases (ESBLs) are chemicals (enzymes) that can be made by some bacteria like E. coli and due to ESBLs any antibiotics become ineffective for treatment due to resistance.

The excessive use of antibiotics in human and livestock resulted in the antibiotic resistance which is not only a threat to public but also a hurdle for the microbiologist and clinicians to prepare treatment plans of livestock diseases (Blaak, 2015, Rupp and Fey, 2003). Resistance of microbes to antibiotics is becoming an alarming situation around the world and the infection caused by such bacteria is much harder to treat. In Pakistan poultry is the largest income producing industry (Pakistan Govt, 2016; Malik et al., 2018). In poultry production system bulk of antibiotics are used not only as growth promoter but also as preventive tool for control of diseases. For prevention and treatment, the use of b-lactam antibiotics makes ESBLs producing bacteria prevalence significantly higher (Dierikx et al., 2013). ESBL-producing bacteria are present almost at each level of the poultry production pyramid and can be detected even in the muconium of the day-old chicks. The surroundings close to poultry shed show high prevalence rate of these bacteria and enduring to the infection pressure with further ESBL-types (Saliu et al., 2017). Pakistan a developing country also suffering from the same threat of antibiotic resistance (Rahman et al., 2019).

Materials and Methods

Study area and collection of samples

The current study to check the prevalence of ESBL E. coli was conducted during September, 2020 to November, 2021. Processing of the samples were conducted in the microbiology section of Veterinary Research and Disease investigation center (VR and DIC) Abbottabad. The sampling was carried out from Abbottabad region and for the purpose of study a total of 210 samples of ceca of freshly slaughtered broilers were collected in sterilized labeled tubes from open market of district Abbottabad. The collected samples were then shifted to the microbiology section of VR and DIC for further laboratory analysis.

Laboratory procedure

One gram of ceca was taken and enriched in 9 ml of peptone water and incubated for 24 hrs at 37oC (Liaqat et al., 2022). After incubation the samples were cultured on Mac-Cef media which was prepared by mixing 1mg/L of cefotaxime sodium in MacConkey agar (Wilson and McCab, 2007; Ullah et al., 2021) and incubated for 24 hs at 37 oC. After incubation for 24 hours the pink colonies were again sub cultured on Mac-Cef media for purification. After purification of the colonies, gram staining and biochemical tests i.e., indole (I), methyl red (MR test), Vogos prosker (VP) test, simmon citerate (SC), triple sugar iron (TSI) were performed using the procedure as mentioned by Harley (Harley and Prescott, 2002).

Drug sensitivity testing

There are many methods available which are practiced in different labs to detect ESBL production in Gram negative bacilli. Among all these methods, the Double Disc Synergy Test (DDST) is complicated and a reliable method (Singh and Singh, 2014). Positive E. coli colonies were further subjected to CDT and DDST tests for detection of ESBL E. coli. The identified E. coli colonies were streaked on Muller Hintin Agar for drug sensitivity (Ullah et al., 2021).

The ESBL production was tested by the Double Disc Synergy Test (DDST) by using a disc of amoxicillin-clavulanate (20/10 μg) along with Ceftizidime. A disc which contained amoxicillin-clavulanate (20/10μg) was placed in the centre of the plate. The discs were placed 15 mm and 20 mm apart respectively, center to center to that of the amoxicillin-clavulanate disc (Paterson and Bonomo, 2005). Any alteration or increase in the zone towards the disc of amoxicillin-clavulanate was considered as positive for the ESBL production (Naseer, et al., 2017; Singh and Singh, 2014; Ullah et al., 2021) as shown in Figure 2.

Results and Discussion

A total 210 samples were streaked on Mac-cef media, 182 samples showed pink colonies and were gram negative (Figure 1 and Table 1). 8 samples were negative (no growth). 20 samples were growth other than E. coli. Our desired work was the isolation of ESBL E. coli.

 

 

Table 1: Culture results.

Total samples	Pink colonies/ E. coli	Yellow colonies	No growth
210	182	20	08

 

The 182 samples were subculture and performed biochemistry.

 

Table 2: ESBL E. coli results.

Total samples	ESBL E. coli positive	Percentage
210	112	53%

 

The 182 samples were subculture and performed biochemistry as shown in Table 2.

 

The CST which showed an inhibition zone size of ≤ 22 mm with antibiotic disc ceftazidime (30 μg) were considered as potential ESBL-producer (ESBL positive) as recommended by CLSI guideline (CLSI, 2014) in combination disc test.

The present research shows that the total isolated samples of ESBL E. coli were 112 through DDST test as shown in Table 2 and 110 through combination disk test.

With the advent of knowledge about the antibiotics and relative phenomenon for the identification of bacteria makes the treatment and control of many diseases possible. But on the other hand, the misuse and overuse of antibiotics in the field has made a challenge and delayed the prognosis. In poultry sector the antibiotics are not only use at the time of disease occurrences but most of the farmers uses the antibiotics regularly in their flocks normal feed and drinking water as preventive measure. In this way the occurrences ratio of diseases was less but this imparts a drastic effect on carcass quality. In this way the introduction of antibiotic resistance occurs. To check the antibiotic resistance and ESBLs E. coli at finished broiler product this study was designed keeping in view the public health importance.

During the current study a total 210 cecal samples collected from the finished broiler product for the market were subjected for isolation of ESBLs E. coli. It was noted that out of 210 samples 08 samples were found negative (no growth of any bacteria found) and 20 samples were shown growth other than E. coli suggesting a total prevalence of E. coli as 86.6 %. The prevalence of ESBLs in Cecum of broilers in the present study is 53% of the total samples tested which is 61% of total positive samples. The results of the current study are in line with the study of Blaak et al. (2015), who has found prevalence of ESBL E. coli as 65% from broilers flocks in Netherland. The results were different to studies of the Rehman et al. (2019) which shown the ESBLs E. coli prevalence’s 47.6 % from chick meat. The results of our study are found higher than Girlich et al. (2007) shown 10.7% prevalence of ESBL E. coli from healthy chicken in France, the difference might be due to the geographical difference, drug legislation and sample difference as they selected meat samples for isolation in France (Girlich et al., 2007). The isolation of ESBL from milk, meat, intestine, and environment may be different because of different habitat. There is the dire need to work on antibiotic resistance throughout the country with special reference to the public health importance.

Conclusions and Recommendation

As per results of the current study the prevalence of ESBLS producing bacteria in poultry is about 50% which is a huge risk to human and livestock. Awareness and counseling of the poultry farmers may be done across the country to stop the use of antibiotics for growth promotion and prevention.

Acknowledgement

The whole laboratory staff specially Imran khan (Lab technician), Hazrat Abubaker (Lab attendant) of Veterinary Research and Disease Investigation Center Abbottabad is highly acknowledged.

Novelty Statement

According to the authers knowledge, the current study is first time initiated in VR& DIC in Abbottabad region, there was no previous data recording the ESBL E.Coli from animal source in the region. The results of this study will definitely help the researchers and students for further studies.

Author’s Contribution

MS gave the idea of this study, MM and ZS performed the lab work, YA and ZA helped in sampling. MM wrote the manuscript. YA and SA also helped in writing the manuscript. Hamidullah supervised the whole lab activities.

Conflict of interest

The authors have declared no conflict of interest.

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