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Identification of the Genotypes of Extended-spectrum β-lactamase Producing Escherichia coli in the Small Intestine of Broiler Chickens at a Traditional Market in Surabaya, Indonesia

Reichan Lisa Az Zahra1, Sasqia Khairunnisa Aulia Prayudi1, Tri Wahyu Suprayogi2, Dhandy Koesoemo Wardhana3, Mustofa Helmi Effendi3,*, Aswin Rafif Khairullah4, Shendy Canadya Kurniawan5
1Profession Program in Veterinary Medicine, Faculty of Veterinary Medicine, Universitas Airlangga. Jl. Dr. Ir. H. Soekarno, Kampus C Mulyorejo, Surabaya 60115, East Java, Indonesia.
2Division of Veterinary Reproduction, Faculty of Veterinary Medicine, Universitas Airlangga. Jl. Dr. Ir. H. Soekarno, Kampus C Mulyorejo, Surabaya 60115, East Java, Indonesia.
3Division of Veterinary Public Health, Faculty of Veterinary Medicine, Universitas Airlangga. Jl. Dr. Ir. H. Soekarno, Kampus C Mulyorejo, Surabaya 60115, East Java, Indonesia.
4Research Center for Veterinary Science, National Research and Innovation Agency, Jl. Raya Bogor Km. 46 Cibinong, Bogor 16911, West Java, Indonesia.
5Master Program of Animal Sciences, Department of Animal Sciences, Specialisation in Molecule Cell and Organ Functioning, Wageningen University and Research, Wageningen 6708 PB, Netherlands.

ABSTRACT

Background: Escherichia coli infection from inside the chicken’s body may be present if the previously slaughtered chicken was contaminated with pathogens or the cage’s hygiene is inadequate. One of the resistance mechanisms against antibiotics used by Gram-negative bacteria of the Enterobacteriaceae family is the production of extended-spectrum β-lactamases (ESBL). Research is required to determine which E. coli genotypes in broiler chicken samples collected from Surabaya’s traditional marketplaces produce ESBL. 

Methods: A total of 100 small intestine samples collected from broiler chickens were used in this study. After initial processing all the samples were separately injected into Eosin Methylene Blue Agar (EMBA) and Indole, Methyl Red, Voges Proskauer and Citrate (IMViC) tests were used to confirm the results. A test for antibiotic sensitivity based on the Kirby-Bauer method called the Double Disk Synergy Test (DDST) was used to analyze isolates that showed resistance to aztreonam and multidrug resistance (MDR). PCR testing was performed on ESBL-verified isolates to detect the TEM and CTX-M genes.

Result: The results of the test for E. coli antibiotic resistance revealed that 14 isolates were proven to be multidrug-resistant (MDR) and six of them were found to be E. coli that produced ESBL. Four isolates were found to carry the TEM and CTX-M genes after genotyping analysis. Understanding the dangers of using antibiotics as feed additives and growth promoters is crucial for farmers to address the problem of ESBL-producing E. coli bacteria in chickens. Furthermore, the government needs to take action to monitor and enforce stronger controls on the use of antibiotics, which are even readily available on the market.

INTRODUCTION

The presence of E. coli contamination which is from inside the chicken’s body can occur if the previously slaughtered chicken has been infected by bacteria, or the sanitary conditions of the cages are poor (Adeyanju and Ishola, 2014). While the slaughtering, handling, air quality and long-term storage of chicken can result in environmental contamination; the small intestine of chicken, in particular, is frequently observed to have this bacterial invasion (Pan and Yu, 2014). Due to the current lack of effective treatments, the survival of bacteria that produce ESBL in food of animal origin may create public health problems like pneumonia, respiratory tract infections, cramping in the stomach, diarrhoea and urinary tract infections in man (Sivakumar et al., 2021).
       
Antibiotic-resistant bacteria can travel through the small intestine of food-producing animals and subsequently be eliminated via feces (Xu et al., 2022). Animal manure-borne resistant bacteria can spread across farms, slaughterhouses, or poultry slaughterhouses, as well as during the meat-processing process (Jeżak and Kozajda, 2022). The environment around farms and slaughterhouses or chicken abattoirs may also be contaminated even though they are far from the source of contamination. This is significant because human-animal disease transmission is possible at any point in the production chain and at any moment (Ramos et al., 2020; Tyasningsih et al., 2022).
       
In Indonesia, people usually purchase chicken at traditional marketplaces because these are the areas where buying and selling activities take place face-to-face and where there is typically a negotiation process (Alfani et al., 2021). Traditional markets are typically associated with filthy, chaotic locations and the chicken that is sold there is typically arranged in a way without any sort of supporting mat to prevent bacterial infection (Siddiky et al., 2022). Surabaya, one of the biggest cities in Indonesia, has several such markets. One of the causes contributing to E. coli contamination is the placement of chicken meat combined with innards in the form of the small intestine (Hussain et al., 2017).
       
Antibiotics can lose their ability to combat E. coli germs over time, leading to uncontrolled infections (Khairullah et al., 2024; Nalband et al., 2020). One of the reasons for this is the inappropriate use of antibiotics to stop the spread of E. coli infection (Aslam et al., 2018; Widodo et al., 2022). Bacterial resistance is more prevalent, making it challenging to treat illnesses, particularly moderate or severe infections (Yunita et al., 2020; Khairullah et al., 2020; Nwobodo et al., 2022). Extended-spectrum β-lactamase (ESBL) synthesis is one of the resistance mechanisms used by Gram-negative bacteria in the Enterobacteriaceae family (Shaikh et al., 2015; Durairajan et al., 2021).
       
This resistance results from acquiring a plasmid with the gene for the ESBL enzyme, which is mostly generated by E. coli (Lemlem et al., 2023). The ability of the enzyme to break down antibiotics like penicillins, cephalosporins and monocyclic amides is crucial for understanding how E. coli develops its antimicrobial resistance, but β-lactamase inhibitors like sulbactam, tazobactam and clavulanic acid typically prevent ESBL production from occurring (Miao et al., 2017). The two primary coding genes for ESBL are TEM and CTX-M (Castanheira et al., 2021). Both of these genes generate ESBL, which hydrolyze β-lactam antibiotics.
       
A particularly severe issue in the field of medicine is bacterial resistance to antibiotics (Khairullah et al., 2023). However, case information on the prevalence of E. coli contamination that produces ESBLs in conventional market contexts is infrequently reported. Research is required to identify the E. coli genotypes that produce ESBL in Surabaya’s traditional marketplaces.

MATERIALS AND METHODS

Study area and sample collection
 
In this investigation, 100 small intestines of broiler chickens from various vendors were collected aseptically from Surabaya City’s traditional markets. This study was carried out from April 2023 to May 2023. This research was conducted at the Department of Veterinary Public Health, Faculty of Veterinary Medicine, Universitas Airlangga, Indonesia.
 
Bacterial isolation of E. coli
 
One gram is the weight of the contents that have been taken out of each chicken’s small intestine. The sample is homogenized using a 0.1% buffered peptone water solution at a ratio of 1:10 as the initial step in isolating E. coli bacteria. The samples were then streaked over sterile loop-adjusted Eosin Methylene Blue Agar (EMBA) at a distance sufficient to divide colony development and incubated for a full day at 37°C.
       
Gram staining was used to identify the colonies and the morphology of E. coli was examined under a 1000x magnification oil immersion microscope. The IMViC tests, which consist of the Indole test, methyl red (MR), Voges Proskauer (VP) test and citrate utilization test, were then used to confirm colonies suspected of being E. coli.
 
Antibiotic resistance of E. coli
 
The isolated and identified pure cultures from physiological saline as a suspension with 0.5 McFarland turbidity (1-2 x 108 CFU/ml) was used for the antibiotic susceptibility test. Using a clean cotton swab, the culture was transferred to Mueller Hinton Agar (MHA), where it was spread out and let to stand for around five minutes. The antibiotic disks were then, using the Kirby-Bauer technique, positioned above the MHA, which had been smeared with pure culture, at a distance of 25-30 mm. After that, the culture was kept at 35°C for 16-18 hours. Five different antibiotics ampicillin (10 µg), gentamicin (10 µg), aztreonam (30 µg), ciprofloxacin (5 µg) and chloramphenicol (30 µg) were used for sensitivity tests and resistance profiling.
 
Tests for antibiotic sensitivity, multidrug resistance (MDR) and resistance to the antibiotic aztreonam were performed on isolates using the Double disk synergy test (DDST). The data were interpreted with the help of the Clinical and Laboratory Standards Institute (CLSI) 2022 guidelines. An antibiotic disk was used to confirm the phenotype. Ceftazidime (30 µg), amoxicillin-clavulanic acid (20 µg) and cefotaxime (30 µg) are among the cephalosporins in this group.
 
Molecular detection of E. coli
 
Five microliters of DNA are utilized as a template. The specific primers utilized were designed to target the TEM and CTX-M genes (Table 1). Mixture for the PCR reaction with a volume of 25 µl consisting of 12.5 µl PCR Mastermix (0.5 U Taq Polymerase, 0.2 mM dNTP, 1.5 mM MgCl2 and Buffer 1x), 1.25 µl for each primer and 5 µl DNA template. The PCR was conducted under the following conditions: initial denaturation at 94°C for 7 minutes, 35 cycles of 96°C for 50 seconds of denaturation, 50°C for 40 seconds of annealing and 72°C for 1 minute of extension and finally 10 minutes of extension at 72°C.
 

Table 1: Primers used in the present study.


       
1.5% agarose was used to visualize the PCR result. Five microliters of the PCR product and one microliter of the marker were mixed together, put on parafilm and left in an ethium bromide solution for ten minutes in the dark. A UV transilluminator with a 360 nm wavelength was used to observe the PCR product that has been produced.

RESULTS AND DISCUSSION

Following morphological culture, Gram staining and biochemical testing, it was shown that 63 (63%) of the 100 intestinal samples collected from broiler chickens tested positive for E. coli (Table 2). The appearance of metallic green bacterial colonies on EMBA media indicated that the morphological culture of E. coli was successfully achieved (Fig 1). A Gram-negative staining result indicated organisms are either pink or red in color and short rods in Gram staining (Fig 2). The appearance of an inverted spruce formation on the SIM test (Motility), an indole ring on the SIM test (Indol positive), a yellow color on the VP test (negative VP), a red color change on the MR test (positive MR) and green on the citrate utilization test (citrate negative) all indicated that the IMViC test had produced positive indications for E. coli (Fig 3).
 

Table 2: Identification of E. coli isolatedfrom faecal samples collected from small intestines of broiler birds.


 

Fig 1: Colonies of E. coli on EMBA.


 

Fig 2: E. coli stained with Gram stain by using normal microscopy.


 

Fig 3: Results of the IMViC tests showed the above positive results indicative for E. coli.


       
The interaction between bacteria and the environment, which infects the chicken’s body through the feed, contributes to the presence of bacteria in the digestive system of chickens (Hossain et al., 2023). Environmental contamination was one of numerous causes of the rise of E. coli bacteria in specific chickens. It is suspected that the chickens that were given a drink before being slaughtered and kept in a place with moist husks containing lots of chicken feces could be a potent source of such infections (Biesek et al., 2023).
       
The E. coli isolates examined in this study showed that 56 showed the highest level of resistance to ampicillin, 19 isolates were resistant to ciprofloxacin, 17 isolates were resistant to gentamicin, 11 isolates were resistant to chloramphenicol and 4 isolates were resistant to aztreonam (Table 3). The E. coli antibiotic resistance test results revealed that of the 14 isolates, 22.22% were confirmed to be multidrug resistant (MDR) due to their resistance to three to five different classes of antibiotics (Fig 4). The most prevalent pattern of antibiotic resistance was found in the isolates that fell into the ampicillin, chloramphenicol, ciprofloxacin and gentamicin) group, which consists of 5 isolates (Table 4).
 

Table 3: Antibiotic group-specific resistance profile of isolated E. coli.


 

Fig 4: The isolates of E. coli showed multidrug resistance in their sensitivity test findings on MHA media.


 

Table 4: Isolates of E. coli with a multidrug resistance profile.


       
Six of the 14 E. coli isolates with the greatest documented resistance pattern to the four antibiotics ampicillin, chloramphenicol, ciprofloxacin and gentamicin were confirmed to be MDR. The development of resistant bacteria demonstrates that these drugs do not stop them from growing and they cannot be treated with them since they have no therapeutic effect (Sandegren, 2014). Bacteria that are classed as intermediates to an antibiotic suggest that the antibiotic has an unreliable therapeutic effect and that its use has started to decline (Breijyeh et al., 2020). The presence of bacteria that are still sensitive to antibiotics indicates that these drugs can still stop bacterial development and have a good chance of having therapeutic effects (Łapińska et al., 2022).
       
The Double Disk Synergy Test was performed on 14 isolates of MDR E. coli and those that had ESBLs identified and subsequently detected using three different antibiotics. Six of the 14 isolates tested by the DDST test were determined to be E. coli strains that produce ESBL (Fig 5). A hazard to the public’s health is posed by the existence of ESBL bacteria that produce MDR (Igbinosa et al., 2023). There may be few effective treatments for certain disorders. Because antibiotics are misused in terms of dosage, diagnosis and infection-causing bacteria, there is a significant incidence of bacterial resistance (Aslam et al., 2021). The use of antibiotics above the recommended dose places a stronger selection pressure on bacteria, causing them to mutate and eventually develop resistance. Antibiotics also have a minimum dose to produce therapeutic effect (Raymond, 2019). Underdosing on antibiotics prevents the bacteria from being entirely eliminated, which forces the remaining germs to adapt and develop antibiotic resistance (Munita and Arias, 2016).
 

Fig 5: Results of the DDST test on isolates of ESBL-producing E. coli.


       
The presence of the TEM and CTX-M genes in six isolates of E. coli that were confirmed to produce ESBLs was subsequently determined by genotyping. Four of the six isolates carried both the TEM and CTX-M genes in the electrophoresis results of the six isolates studied, while the other two isolates contained just the CTX-M gene (Table 5). The emergence of a single band on the electrophoresis findings indicates a favorable outcome (Fig 6).
 

Table 5: TEM and CTX-M isolate E. coli gene molecular identification.


 
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The results of the ESBL resistance gene detection from the six isolates under examination revealed that four of the isolates had both the TEM and CTX-M genes, while the other two isolates had only the CTX-M gene. The β-lactamase enzyme is typically to blame for the presence of these two genes in Gram-negative bacteria (Tooke et al., 2019). The plasmids that contain the antibiotic resistance genes for aminoglycosides, trimethoprim, sulfonamides, tetracyclines and chloramphenicol have ESBL that can hydrolyze antibiotics (Wibisono et al., 2021). The plasmid also harbors the TEM and CTX-M genes, which are descended from the bacterial chromosome (Faridah et al., 2023; Widodo et al., 2023). Integrins and transposons influence gene activity.
       
The two primary categories of β-lactam antibiotics are TEM and CTX-M genes (Bajpai et al., 2017). However, certain studies have reported a higher prevalence of the CTX-M gene (Leão et al., 2021; Abo-Almagd et al., 2023; Seo and Lee, 2021). The ability of the bacteria to resist antibiotics is due to the presence of a detectable antibiotic resistance gene (Kunhikannan et al., 2021). This is suggested by the existence of genes found in isolates that show resistance in the sensitivity test. The phenomena where Gram-negative bacteria can transmit horizontal or lateral gene transfer from plasmids and clone ESBL-producing E. coli present in animal bodies is linked to the high prevalence of CTX-M and TEM in creating E. coli resistance to third-generation cephalosporin antibiotics (Chukwu et al., 2022; Mondal et al., 2022).
       
The key to resolving the issue of ESBL-producing E. coli bacteria in chickens is for farmers to be aware of the risks of using antibiotics as feed additives and growth promoters (Saliu et al., 2020; Sakthikarthikeyan et al., 2023). In addition, constant use of disinfectants to keep the cage clean is necessary (White et al., 2018). Furthermore, the government needs to take action to monitor and enforce stronger controls on the use of antibiotics, which are even readily available on the market.
       
Maintaining a clean environment and washing hands before eating are two ways to prevent foodborne illness (Khairullah et al., 2022; Ramandinianto et al., 2020). Because many workers in traditional markets are unaware of the risks associated with slaughtering poultry without personal protective equipment, they must wear work safety equipment like masks, gloves and boots as a preventative measure to avoid being exposed to bacteria that are resistant to multiple antibiotics (Verbeek et al., 2020).

CONCLUSION

To conclude the study it can be said that 14 MDR isolates were isolated from 100 chicken intestine samples, 6 of which produced ESBL and 4 of which had the TEM and CTX-M genes. The key to resolving the issue of ESBL-producing E. coli bacteria in chickens is for farmers to be aware of the risks of using antibiotics as feed additives and growth promoters.

ACKNOWLEDGEMENT

This study was supported in part with the Penelitian Unggulan Airlangga (PUA) Universitas Airlangga, Indonesia, in the fiscal year 2023, with grant number: 1710/UN3.LPPM/PT.01.03/2023.

CONFLICT OF INTEREST

The authors have declared no conflict of interest.

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