Prevalence of multidrug-resistant and extended-spectrum β-lactamase producing Escherichia coli from local and broiler chickens at Cibinong market, West Java, Indonesia

Background and Aim: Antimicrobial resistance (AMR) is becoming a public health concern. Foodborne pathogens are infectious agents that can be transmitted from animals to humans through food and can become resistant due to misuse and overuse of antibiotics, especially in poultry. This study aimed to detect the prevalence of multidrug-resistant and extended-spectrum β-lactamase (ESBL)-producing Escherichia coli isolated from local and broiler chickens at the Cibinong market, West Java, Indonesia. Materials and Methods: A total of 60 cloacal swab samples from 30 local and broiler chickens sold at the Cibinong market in West Java were obtained by random sampling. From these samples, 39 E. coli isolates were obtained after being cultured on eosin methylene blue agar and molecularly identified using polymerase chain reaction (PCR). Six antibiotic disks were used for the antibiotic sensitivity test against E. coli isolates cultured on Mueller-Hinton agar. PCR was performed to detect ESBL genes (blaTEM, blaSHV, and blaCTX-M). Results: A total of 76.47% (39/51) cloacal swab samples were positive for E. coli. All E. coli isolates were sensitive to imipenem (100%), and 38 isolates were sensitive to cefoxitin (FOX) (97.4%). On average, the isolates were sensitive to amoxicillin-clavulanic acid (AMC) (69.2%) and ceftriaxone (CRO) (89.7%). E. coli isolates were occasionally resistant to enrofloxacin (25.64%), followed by gentamicin (20.51%), CRO (10.25%), AMC (7.69%), and FOX (2.56%). The prevalence of E. coli AMR was 10.25% (4/39). All four multidrug-resistant E. coli isolates (blaTEM and blaCTX-M) were confirmed to have the ESBL gene based on PCR. Conclusion: The prevalence of multidrug-resistant and ESBL-producing E. coli is still found, proving that there is still inappropriate use of antibiotics and a need for strict supervision of their use, especially around Cibinong market, West Java.


Introduction
Antibiotics commonly used in human medicine and animal husbandry have increased antibiotic resistance, resulting in global health problems [1].It is caused by misuse and overuse of antibiotics [2].The World Health Organization (WHO) has identified antibiotic resistance as one of the top health risks of the 21 st century, with approximately 1.2 million people killed each year, especially in developing countries, including Indonesia [3,4].Poultry farms contribute to antibiotic resistance because inappropriate use of antibiotics is often found in chicken farms [5].Antibiotics are usually used for treating and preventing bacterial infections and growth boosters, especially in poultry production [5].Although antibiotic growth promoters have been banned by the Indonesian government, farmers continue to use them because of the low level of supervision by the authorities [6].Antibioticresistant bacteria may indirectly reach humans along the food chain through the consumption of contaminated food or food-derived products and following direct contact with colonized/infected animals or biological substances, such as blood, urine, feces, saliva, and semen [7].In addition, if antibiotic-resistant bacteria in the human body lead to infection and spread to other people, public health problems may occur.As a result, the treatment and healing process with certain antibiotics becomes ineffective [8].
Indonesia has a higher antimicrobial resistance (AMR) level than Australia and New Zealand [9,10].
Furthermore, according to the global AMR surveillance system 2021 report by the WHO [11], there was an increase in AMR bloodstream infections (BSIs) of Escherichia coli and Salmonella spp. with an increased rate of 15% compared to 2017 in 99 countries, territories, and regions.Several types of pathogenic bacteria cause BSIs, one of which is E. coli [11].The presence of E. coli is a sign of hygiene and sanitation.Cibinong Market, located in Bogor Regency, West Java, is a traditional wet market that sells various goods such as vegetables, fruits, fish, meat, and live birds.Livestock poultry, such as broiler chickens and local chickens imported from different areas, are sold there.As a result, assessing zoonotic E. coli AMR in the Cibinong market will help us understand the state of antibiotic contamination in the animal environment and serve as a reference for developing livestock husbandry rules and conserving the natural environment in Bogor Regency.
This study aimed to determine the presence of antibiotic-resistant and extended-spectrum β-lactamase (ESBL) E. coli in local and broiler chicken sold at the Cibinong market to ensure public health.

Ethical approval
Ethical approval was not necessary for this study.Cloacal swab samples were collected shortly after the seller sold the chickens.However, samples were collected aseptically in accordance with established collection methods.
Using a collection swab (Citoswab™, Jiangsu, China), samples of cloacal swabs were randomly taken from 30 local chickens and 30 broilers sold at the Cibinong market.After the samples were aseptically collected, they were immediately sent to the InaCC for bacteriological analysis.Citoswab™ was cultured on eosin methylene blue agar (EMBA, Himedia™, Mumbai, India) and incubated at 37°C for 24-48 h to identify the E. coli strains.After the incubation period, a green metallic sheen with black center colonies was assumed to be E. coli.Therefore, molecular confirmation using polymerase chain reaction (PCR) by uspA gene detection is necessary for identification [12].

Antimicrobial susceptibility test
Confirmed E. coli isolates were subsequently cultured on Mueller-Hinton agar (MHA, Oxoid™, Basingstoke, England) subsequently.A bacterial suspension with turbidity equal to the 0.5 McFarland standard was readied and evenly spread on (MHA, Oxoid™).Antibiotic disks were placed on the agar surface.Subsequently, it was incubated at 37°C for 16-18 h.Amoxicillin-clavulanic acid (AMC, 30 µg), ceftriaxone (CRO, 30 µg), cefoxitin (FOX, 30 µg), imipenem (IPM, 10 µg), enrofloxacin (ENR, 5 µg), and gentamicin (CN, 10 µg) were used as antibiotic disks.The antimicrobial sensitivity test was performed according to the recommendations of the Clinical Laboratory Standards Institute [13].We examined the inhibitory zone and measured its diameter using a caliper.E. coli ATCC 25922, considered as the positive control, and Staphylococcus aureus ATCC 25923, designated as the negative control, were employed to evaluate the susceptibility classification (sensitive, intermediate, or resistant).

ESBL gene detection
The multidrug-resistant E. coli isolates were then tested for resistance genes blaTEM, blaCTX-M, and blaSHV using a polymerase chain reaction [12,14].Table-1 shows the sequences of the oligonucleotide primers and the size of the PCR products.PCR was performed in a total volume of 30 µL consisting of 1.5 µL for each forward and reverse primer, 2 µL DNA template, 15 µL MyTaq HS red mix (Bioline GmbH, Germany), and 10 µL nuclease-free water (Thermo Fisher Scientific™, USA).

Statistical analysis
The data are presented descriptively in percentages displayed in tables.

Results
In the present study, 51 isolates were isolated from broiler chickens and local chickens on EMBA.From these isolates, 39 samples were identified as E. coli based on the results of the PCR assay by detecting uspA gene sequences (Table-1) [12,14,15].

Sensitivity of E. coli to various antibiotics revealed
Table-1: Primers used for the detection of Escherichia coli and ESBL genes.

Gene
Primer sequences Size (bp) Reference  -3 and 4).MDR is defined as acquired resistance to at least one bacterial agent for three or more antibiotic groups [16].

Discussion
Poultry is one of the most important sources of food in the world.Antimicrobial-resistant E. coli can be transmitted to humans from contaminated poultry products or by contact with poultry waste [17].A total of 60 cloacal swabs from 30 broilers and 30 local chickens from Cibinong market, West Java, Indonesia were used as samples.Swabs were cultured on EMBA.The colony morphology of 51 isolates showed the growth of green metallic sheen with black centers, presumptively indicative of E. coli (Figure-1) [18].All isolates were confirmed using PCR for molecular typing of the uspA target gene.Only 39 samples (76.5%) were identified as E. coli.These results are similar to those reported by Chen and Griffiths [19] and Godambe et al. [20], who used the uspA gene as a DNA marker for the identification of E. coli (Figure -2).In a previous study in Indonesia, the uspA gene was also used to identify E. coli from broilers [21].The antimicrobial sensitivity test was performed on isolates identified as E. coli using the disk diffusion method.This study showed that E. coli had the highest resistance level against ENR, followed by CN and CRO, while AMC and FOX had much lower resistance levels.These results are consistent with several studies on antibiotic resistance in E. coli from chickens in Indonesia.Hardiati et al. [21] reported that E. coli from broilers are resistant to ENR and CN.The resistance of E. coli to ENR and CN was also similar to that reported in studies using broiler samples in Indonesia [22,23].Abo-Amer et al. [17] reported that E. coli isolated from chicken cloacae in Saudi Arabia were resistant

Sample code ESBL gene blaTEM blaCTX-M blaSHV
to CN, AMC, and CRO; however, the resistance levels of CRO and AMC in this study were much lower than those reported by Lemlem et al. [24].According to Ali et al. [25], E. coli isolated from chicken meats were resistant to CN but had a lower percentage of resistance to ENR and AMC.Aworh et al. [26] also reported resistance against CN and FOX in chickens.
Resistance to AMC and CRO may be used to determine the activity of ESBL enzyme.In addition, there is an increased risk of infection with ESBL-producing bacteria associated with second-and third-generation cephalosporins [27,28].This study revealed that four isolates (10.2%) of E. coli from broilers were MDR (Figure -3), with three isolates resistant to four groups of antibiotics and one isolate resistant to three antibiotic groups.There have been many reports of AMR in broilers worldwide.This may be due to the overuse of antibiotics for disease prevention, to reduce bacterial invasion control, and as growth promoters to increase poultry production [22,28].The MDR observed in this study could be mediated by mobile genetic components, such as resistance genes [17].Antimicrobials continue to be used outside the health-care system for various purposes and play an important role in animal production, particularly for veterinary and livestock purposes [29].Furthermore, MDR is caused by uncontrolled antibiotic use, leading resistant bacteria to evolve through genetic mutations, swap genetic materials, and proliferation to fight against various antibiotics [30].Antimicrobial-resistant E. coli may remain in the intestinal tracts of poultry for long periods, regardless of the use of antimicrobials, and can serve as a route for transmission to the human population by consumption [17].
Four MDR E. coli isolates were positive for the ESBL-encoding genes blaCTX-M and blaTEM, but negative for blaSHV.According to a study conducted by Lukman et al. [31], ESBL-producing E. coli was present in broiler feces in Bogor, Indonesia, which was related to the findings in this study.The results of this study are in accordance with several studies in Indonesia which reported that the ESBL-producing E. coli were identified from feces of broilers in Bogor reached 25% [32]; cloacal swabs of broilers from wet markets in Surabaya [23]; cloacal swab of layers in Blitar reached 7.03% [28]; swab cages and wastewater around broiler farm in Pasuruan 9.14% [33]; samples of chicken meat sold at Purwokerto's market reached 7.14% [34]; from environmental samples of dairy farms in Yogyakarta, Probolinggo, and East Java [35][36][37]; and from companion dogs in Surabaya reached 9.41% [38].These findings were also similar to those reported by Lemlem et al. [24], who detected blaCTX-M and blaTEM from broilers in Malaysia.Gundran et al. [39] also reported the detection of blaCTX-M and blaTEM in broilers from the Philippines.Numerous studies have reported that the same isolates have multiple beta-lactamase genes [38,39].Recently, blaCTX-M was identified as the leading ESBL gene in the world [24,39].The most commonly detected ESBL gene was blaCTX-M, followed by blaTEM and blaSHV [40].The prevalence of ESBLs varies throughout the world, with Asian countries having the highest rates [41].The results of this study suggest that local broiler farms that sell broilers at the Cibinong market are a source of ESBL-producing E. coli.The spread of ESBL-producing E. coli isolates from food animals without symptoms suggests that commensal E. coli may function as a reservoir for resistance genes and may constitute a danger of human transmission [39,42].
ESBL-producing E. coli in this study showed that the prevalence of ESBL-producing E. coli in Indonesia is also increasing.A zoonotic disease may be caused by ESBL-producing bacteria in the environment, which may spread from animals to humans.Direct contact with the contaminated environment and consumption of contaminated meat are two ways to spread the disease.ESBL-producing E. coli can contaminate chicken meat if it is handled unhygienically during slaughter and handling if the chickens have the bacteria in their digestive tracts, or if it originates from a contaminated cage environment [33].Several age groups, including pregnant women, children, the elderly, those with immunosuppression, as well as post-operative and chemotherapy patients, have a high potential for resistance to ESBL-producing E. coli [43].ESBL-producing E. coli causes increased morbidity and mortality [15].

Conclusion
The prevalence of MDR in E. coli isolates from broiler chickens and the discovery of ESBL genes (blaTEM and blaCTX-M) in the present study indicate that antibiotics are used more often in broiler chickens than in local chickens.Therefore, the use of antibiotics should be regulated and monitored regularly.

Figure- 3 :
Figure-3: The antimicrobial susceptibility test result for Escherichia coli was performed on Mueller Hinton agar.
Results of antibiotic sensitivity test for 39 isolates of Escherichia coli.

Table - 3
: The results of antimicrobial resistance isolates based on chicken type.

Table - 4
: The molecular detection results of ESBL gene.