Open Veterinary Journal, (2025), Vol. 15(1): 92-97

Research Article

10.5455/OVJ.2025.v15.i1.8

Molecular detection of blaTEM-encoding genes in multidrug-resistant Escherichia coli from cloacal swabs of ducks in Indonesia farms

Susmitha Nur Ahadini¹, Wiwiek Tyasningsih^2*, Mustofa Helmi Effendi³, Aswin Rafif Khairullah⁴, Muhammad Khaliim Jati Kusala⁴, Ima Fauziah⁴, Latifah Latifah⁵, Ikechukwu Benjamin Moses⁶, Sheila Marty Yanestria⁷, Kartika Afrida Fauzia^8,9, Dea Anita Ariani Kurniasih¹⁰ and Syahputra Wibowo¹¹

¹Master Program of Veterinary Science and Public Health, Faculty of Veterinary Medicine, Universitas Airlangga, Surabaya, Indonesia

²Division of Veterinary Microbiology, Faculty of Veterinary Medicine, Universitas Airlangga, Surabaya, Indonesia

³Division of Veterinary Public Health, Faculty of Veterinary Medicine, Universitas Airlangga, Surabaya, Indonesia

⁴Research Center for Veterinary Science, National Research and Innovation Agency (BRIN), Bogor, Indonesia

⁵Research Center for Animal Husbandry, National Research and Innovation Agency (BRIN), Bogor, Indonesia

⁶Department of Applied Microbiology, Faculty of Science, Ebonyi State University, Abakaliki, Nigeria

⁷Faculty of Veterinary Medicine, Universitas Wijaya Kusuma Surabaya, Surabaya, Indonesia

⁸Research Center for Preclinical and Clinical Medicine, National Research and Innovation Agency (BRIN), Bogor, Indonesia

⁹Department of Environmental and Preventive Medicine, Faculty of Medicine, Oita University, Yufu, Japan

¹⁰Research Center for Public Health and Nutrition, National Research and Innovation Agency (BRIN), Bogor, Indonesia

¹¹Eijkman Research Center for Molecular Biology, National Research and Innovation Agency (BRIN), Bogor, Indonesia

*Corresponding Author: Wiwiek Tyasningsih. Division of Veterinary Microbiology, Faculty of Veterinary Medicine, Universitas Airlangga, Surabaya, Indonesia. Email: wiwiek-t [at] fkh.unair.ac.id

Submitted: 20/09/2024 Accepted: 02/12/2024 Published: 31/01/2025

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Abstract

Background: Globally, there is an increasing frequency of community-acquired illnesses caused by extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli. The presence of ESBL-producing E. coli in livestock is a concern, considering its transmission potential to humans, effects on animal health, risks to food safety, and the widespread spread of antibiotic resistance in both human and animal populations.

Aim: This study investigated the prevalence and characterization of ESBL-producing E. coli in cloacal swab samples collected from duck farms in Jombang, Indonesia.

Methods: In total, 125 cloacal swab samples of ducks were collected from farms. Samples were processed and analyzed for E. coli isolation using standard microbiology techniques. Isolated E. coli strains were further subjected to antimicrobial susceptibility testing and ESBL phenotypic detection using disc diffusion and double-disk synergy test techniques, respectively. Identified multidrug-resistant (MDR) E. coli strains were thereafter screened for the detection of blaTEM ESBL gene by PCR.

Results: A total of 94 (52.2%) out of the collected 180 swab samples were positive for E. coli. Twenty-five (39.1%) out of the recovered E. coli isolates were generally noted to exhibit MDR traits. Exactly 24 (96%) out of the 25 MDR E. coli strains that were selected for molecular studies harbored the blaTEM gene.

Conclusion: The detection of MDR E. coli harboring blaTEM ESBL gene in ducks in our study area is a significant public health problem. Therefore, strong and impactful preventive measures, which would curtail the increasing dissemination of MDR bacterial pathogens in agricultural settings, are urgently needed.

Keywords: blaTEM gene, ducks, E. coli, multidrug resistance, public health, antibiotics.

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Introduction

The Indonesian population is increasing every year, causing the need for food to increase. Duck, as a source of animal protein, has become a food choice for people because not only does it taste delicious but also it is quite affordable; therefore, more duck farms have emerged to meet market needs (Kuźniacka et al., 2020). There are many openings of new farms that have a positive impact on meeting people’s food needs, but livestock also have a negative impact on the form of waste, which has the potential to cause environmental pollution (Godde et al., 2021; Riwu et al., 2022).

Based on data from the National Statistics Agency (CBS, 2019), the duck population in Jombang Regency in 2019 was 2,85,176, which has increased to 3,15,822 in 2020; this number is above the average number of duck populations in other cities and districts in East Java. The number of ducks in Jombang Regency continues to increase, indicating that many new farms have opened in this area, where it could be said that the duck farming business in Jombang Regency has the potential to grow even bigger. One of the livestock wastes is livestock manure. Coliform bacteria are commonly found in feces (Alegbeleye and Sant’Ana, 2020).

Coliform bacteria consist of four major genera, namely Enterobacter, Klebsiella, Citrobacter, and Escherichia. Escherichia coli is one of the most commonly found groups of coliform bacteria. There are E. coli strains that are commensal, harmless, and some that are pathogenic in humans and animals (Ramos et al., 2020). E. coli in poultry can cause diseases, such as colibacillosis, omphalitis, air sacculitis, peritonitis, and salphingitis (Effendi et al., 2022a). The use of antibiotics in commercial livestock can be said to be unavoidable because the large population of livestock in a limited space makes the possibility of disease occurring in a pen greater and giving antibiotics is one of the preventive measures that is usually taken (Tyasningsih et al., 2022). Inappropriate administration of antibiotics can result in antibiotic resistance, which can be detrimental to life. Gram-negative bacteria worldwide continue to develop resistance to β-lactam antibiotics due to the widespread use of these antibiotics as the first-line treatment for bacterial illnesses (Putri et al., 2023).

The persistent exposure of β-lactams to bacteria leads to continuous production of β-lactamase mutations that expand activity against even the newest β-lactam antibiotics. This enzyme is known as extended-spectrum β-lactamase (ESBL) (Wibisono et al., 2021). Resistance resulting from ESBL often coexists with resistance to various antibiotic families that are often employed in human treatment. Since ESBL-producing E. coli bacteria can be transmitted to people, they pose a risk to animal health, food safety, and the spread of antibiotic resistance in both human and animal populations (Yanestria et al., 2022). ESBL-producing bacteria in humans cause limited treatment options because the bacteria usually exhibit resistance to many classes of antibiotics. Urinary tract infection (UTI) in humans is a bacterial infection that does not have many antibiotic options for treatment because it is mostly caused by ESBL-producing E. coli (Zhou et al., 2023). Research on ESBL-producing bacteria is needed within the scope of veterinary medicine, which concerns public health. Knowing the spread of ESBL-producing bacteria in animals is necessary to develop strategies to mitigate its spread.

Although there are a series of reports on the spread of ESBL-producing E. coli in livestock, especially chickens, and cattle, among others; however, studies conducted on ducks, which are also one of the major sources of animal protein in Indonesia, are scarce, hence, this study.

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Materials and Methods

Ethical approval

Ethical clearance for this study was approved by the Faculty of Veterinary Medicine, Universitas Airlangga, Indonesia’s ethical committee on the use of animals in research (Ethics no: 1.KEH.073.05.2024).

Collection of samples

A total of 180 cloacal swab samples of ducks were aseptically collected in three distinct duck farms located in Jombang, East Java, Indonesia, between March 2024 and July 2024. Collected cloacal swab samples were labeled accordingly and transported to the laboratory within two hours of collection for bacteriological analysis.

Bacteriological analysis of samples

Collected duck cloacal swabs were first enriched in MacConkey broth (Merck, Germany) and incubated at 37˚C for 24 hours. A loopful from the inoculated MacConkey broth was then transferred to eosine methylene blue (EMB) agar (Merck, Germany) plates and incubated at 37˚C for 24 hours. Colonies typical of E. coli (greenish-metallic sheen on EMB agar) were subcultured through successive streaking in order to obtain pure colonies, which were then subjected to Gram staining and further biochemical characterization such as IMViC (indole, methyl red, Voges–Proskauer, and citrate tests). Pure colonies of identified isolates were then preserved at 4°C for further tests.

Antimicrobial susceptibility testing and phenotypic ESBL detection

This was done by the disc diffusion method on Mueller–Hinton agar (MHA) according to the Clinical and Laboratory Standards Institute (CLSI) guidelines (CLSI, 2020). Mueller–Hinton (MH) agar (Merck, Germany) was prepared according to the manufacturer’s instructions. An 18–24-hour-old broth culture of the test isolate was standardized to 0.5 McFarland’s standard. A sterile swab stick was inserted into the standardized inoculums and inoculated by spreading on the surface of the prepared MHA plate. The inoculated MHA plate was allowed to dry for a few minutes at room temperature with the lid closed. The following antimicrobials (Oxoid, UK) were tested against the isolates: ampicillin (10 µg), erythromycin (15 µg), tetracycline (30 μg), ciprofloxacin (5 µg), and aztreonam (30 µg). All antibiotic discs were carefully placed on the inoculated MHA plates using sterile forceps. The inoculated MHA plates were then incubated at 37°C for 18–24 h. After incubation, the CLSI recommendations were followed in measuring, recording, and interpreting inhibition zone diameters as resistant or susceptible (CLSI, 2020). E. coli ATCC 25923 and Klebsiella pneumoniae ATCC 700603 were used as quality control strains. Phenotypic detection of ESBL-producing E. coli was done using the double-disk synergy test as previously described (Wibisono et al., 2021).

blaTEM gene detection

All the 25 isolated strains that were identified as MDR were further subjected to molecular characterization for blaTEM gene using PCR. First, bacterial DNA was isolated using the QIAamp^® DNA Mini Kit (QIAGEN, Germany) according to the manufacturer’s protocol. The primers used in this study were blaTEM-Forward 5’-ATGAGTATTCAACATTTCCG-3’ and blaTEM-Reverse 5’-CTGACAGTTACCAATGCTTA-3’ (Ballhausen et al., 2014). The PCR reaction conditions were performed as previously described (Fouladi et al., 2011) in a thermocycler with slight modifications, which included pre-denaturation for one minute at 95°C, 40 denaturation cycles for one minute at 95°C, annealing for one minute at 55°C, and extension for one minute at 72°C. A final extension lasting two minutes at 72°C marked the end of the amplification step. Electrophoresis was run in a 2% agarose gel with SYBR^® safe DNA gel stain at 100 V and 250 mA for 35 minutes. A UV transilluminator was used to view the amplified PCR products.

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Results

Results showed that 94 (52.2%) out of the 180 cloacal swab samples collected from ducks in duck farms located in Jombang, East Java, Indonesia were positive for E. coli. Isolates were noted to be green metallic sheen colonies in EMB agar. They were Gram-negative and were positive for motility, indole, and methyl red tests but were negative for hydrogen sulfide production, Voges–Proskauer (VP), and citrate tests.

Isolates were also observed to be resistant to aztreonam (98.9%), erythromycin (94.7%), tetracycline (57.4%), ampicillin (41.5%), and ciprofloxacin (12.8%) being the least (Table 1, Fig. 1). Twenty-five (39.1 %) out of the recovered E. coli isolates were generally noted to exhibit MDR traits. None of the E. coli strains displayed ESBL production via the double-disk synergy test (DDST); however, 24 (96%) out of the 25 MDR E. coli strains that were selected for molecular studies harbored blaTEM ESBL gene (Figs. 2 and 3).

Table 1. Antimicrobial susceptibility profiles of E. coli recovered from cloacal swabs of ducks

Fig. 1. Representative picture of antimicrobial susceptibility testing of E. coli isolates.

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Discussion

Bacterial pathogens, such as E. coli, which belongs to the Enterobacteriaceae family and harbors the extended-spectrum beta-lactamase (ESBL)-encoding genes, are one of the main contributors to the challenging public health problems in the world today (WHO, 2016). Bacteria that harbor the ESBL genes can produce ESBL enzymes, which can hydrolyze beta-lactam antibiotics, including third- and fourth-generation cephalosporins. Many of the identified ESBL gene variants, including blaTEM, blaSHV, and blaCTX-M, have been identified to be plasmid-mediated (Husna et al., 2023).

In this study, 94 (52.2%) E. coli were recovered from 180 cloacal swab samples of ducks in various duck farms in Jombang, East Java, Indonesia, with only 25 being identified as exhibiting MDR strains. All the isolates were negative for the phenotypic detection of ESBL using the DDST technique; however, 24 out of the 25 MDR E. coli strains that were further molecularly characterized harbored the blaTEM gene.

DDST is a simple and easy method for detecting ESBL, but DDST does not detect all ESBL-producing isolates. Additionally, the CLSI recommends a phenotypic confirmatory disk diffusion test (PCDDT) as a confirmation test for ESBL. However, we suspect that the non-detection of ESBL enzymes in the E. coli strains in this study by the DDST test might possibly be due to the production of Chromosomal cephalosporinase (Kaur et al., 2013). The results of our study have further reinforced that DDST needs to be followed up with a PCR for the genetic confirmation of ESBL-production, thus, making PCR a gold standard for ESBL detection.

Fig. 2. PCR result for blaTEM detection in E. coli isolates (1/2).

Fig. 3. PCR result for blaTEM detection in E. coli isolates (2/2).

The frequency of blaTEM gene (96%) among the 25 MDR E. coli isolates that were molecularly characterized in this study is higher than the study of Sarker et al. (2023) who also reported blaTEM gene in 72.4% of duck cloacal swab samples.

Inappropriate and excessive use of antibiotics cause selective pressure that favors the growth of resistant bacteria. Through conjugative plasmid-mediated horizontal gene transfer, the colonization of resistant bacteria in the intestines of people and animals results in the transmission of bacterial resistance genes in the intestinal flora. The majority of plasmid genes encode ESBLs. It was discovered that Enterobacteriaceae possessed plasmids containing resistance genes. The blaTEM gene is located on a plasmid (Gulfraz et al., 2014). The IncFII plasmid group is known to be a group that encodes ESBL, is widely distributed in Enterobacteriaceae, and is called the epidemic resistance plasmid group. blaTEM is one of the dominant ESBL genes in the world and is found in human, animal, and environmental isolates. The blaTEM gene was found mainly in E. coli and Klebsiella pneumoniae bacterial isolates (Soekoyo et al., 2020). The blaTEM gene is a beta-lactamase most often found in Gram-negative bacteria. Most ampicillin resistance in E. coli is caused by the presence of the blaTEM gene (Effendi et al., 2022b). The existence of resistant bacteria in food-producing animals is caused by the continuous use of antibiotics for therapeutic and non-therapeutic purposes in production systems, which may be found in products of animal origin.

It is known that the main mechanism by which microbial populations can survive in threatened situations is through genetic mutation, expression of a latent resistance gene, or genes that have resistance determinants. Some bacteria have extrachromosomal genes in the form of plasmids or bacteriophages. The resistance factor that is transferred from a chromosome to a plasmid or on a transposon or integron is an R factor plasmid, which is called an infectious plasmid. This transfer can occur through conjugation, transformation, or transduction. Transposons containing resistance genes carry out inversion into plasmids so that resistance genes can be transferred to other cells (Partridge et al., 2018).

The blaTEM resistance gene harbored by the E. coli strains in this study may be plasmid-mediated or chromosomal-mediated. However, a limitation of our study was our inability to carry out further test to determine the location of the blaTEM resistance gene in the recovered E. coli isolates from duck cloacal swabs.

In a study conducted by Oberoi et al. (2013), 96 (35.16%) ESBL-producing Gram-negative bacterial pathogens were also reported, out of which 45.9% were implicated as culprits in UTIs. Additionally, the largest ESBL producers in the study of Oberoi et al. (2013) were Escherichia coli (56.25%), Pseudomonas aeruginosa (18.75%), and Klebsiella pneumoniae (15.62%).

Infections caused by MDR Gram-negative bacteria, which produce various types of β-lactamase enzymes, affect the length of stay in the ICU, as they are associated with patient morbidity and mortality. Many β-lactamases are encoded by chromosomal or transferable genes, located on plasmids or transposons. Initially, the β-lactamase enzyme was most often found in Klebsiella sp. and E. coli, but now, this enzyme is produced by all members of the Enterobacteriaceae and other Gram-negative bacilli (Al-Sheboul et al., 2023).

blaTEM is one of the common beta-lactamase genes found in ESBL-producing Escherichia coli. It was one of the earliest beta-lactamase genes identified and is widely distributed among Enterobacteriaceae, including E. coli. The presence of E. coli encoding ESBL genes in poultry, including ducks, has been documented globally, including in Southeast Asia where poultry farming is prevalent.

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Conclusion

Our study has shown that ducks, which are important sources of animal protein in East Java, Indonesia, are colonized by MDR E. coli, which also harbors blaTEM ESBL gene. The detection of these pathogenic MDR E. coli strains in duck farms in our study area is a significant public health problem, which requires urgent attention. It is therefore imperative to establish strong guidelines and control measures, which would curtail the increasing spread of MDR bacterial pathogens in agricultural settings, especially in duck farms.

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Acknowledgments

This study was done to fulfill the requirements for the degree in Masters of Science in Veterinary Disease and Veterinary Public Health, Faculty of Veterinary Medicine, Universitas Airlangga, Indonesia.

Conflict of interest

The authors declare that there is no conflict of interest.

Funding

This study was partly supported by the International Research Consortium, Lembaga Penelitian dan Pengabdian Masyarakat, Universitas Airlangga, Surabaya, Indonesia Year 2024 with grant number: 171/UN3.LPPM/PT.01.03/2024.

Author’s contributions

SNA and WT: Conceptualization and design. IF, LL, and MKJK: Acquisition of data. MHE and IBM: Formal analysis and interpretation of data. ARK and KAF: Writing-original draft preparation. SMY, DAAK, and SW: Writing-review and editing. All authors have read and agreed to the published version of the manuscript.

Data availability

All data are available in the manuscript.

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