Open Veterinary Journal, (2026), Vol. 16(5): 2849-2859

Research Article

10.5455/OVJ.2026.v16.i5.27

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Molecular detection of CTX-M ESBL–encoding genes in Escherichia coli isolated from quail cloacal swabs in Surabaya markets

Wiwiek Tyasningsih^1*, Maria Oliva Keytimu², Aswin Rafif Khairullah³, Freshinta Jellia Wibisono⁴, Ummi Rahayu², Mustofa Helmi Effendi^5,6, John Yew Huat Tang⁶, Mariana Febrilianti Resilinda Putri⁷, Irfan Alias Kendek⁸, Riza Zainuddin Ahmad³, Bima Putra Pratama⁹, Saifur Rehman¹⁰, Wasito Wasito³, Dea Anita Ariani Kurniasih¹¹ and Ilma Fauziah Ma'ruf¹²

¹Division of Veterinary Microbiology, Faculty of Veterinary Medicine, Universitas Airlangga, Surabaya, Indonesia

²Master Program of Veterinary Science and Public Health, Faculty of Veterinary Medicine, Universitas Airlangga, Surabaya, Indonesia

³Research Center for Veterinary Science, National Research and Innovation Agency (BRIN), Bogor, Indonesia

⁴Department of Veterinary Public Health, Faculty of Veterinary Medicine, Universitas Wijaya Kusuma Surabaya, Surabaya, Indonesia

⁵Division of Veterinary Public Health, Faculty of Veterinary Medicine, Universitas Airlangga, Surabaya, Indonesia

⁶School of Food Industry, Faculty of Bioresources and Food Industry, Universiti Sultan Zainal Abidin (Besut Campus), Besut, Malaysia

⁷Department of Anatomy, Physiology, Pharmacology, Biochemistry (AFFB), Faculty of Medicine and Veterinary Medicine, Universitas Nusa Cendana, Kupang, Indonesia

⁸Department of Microbiology, Faculty of Health, Pharmacy Study Program, Universitas Sari Mulia, Banjarmasin, Indonesia

⁹Research Center for Process Technology, National Research and Innovation Agency (BRIN), South Tangerang, Indonesia

¹⁰Department of Pathobiology, Faculty of Veterinary and Animal Sciences, Gomal University, Indus HWY, Dera Ismail Khan, Pakistan

¹¹Research Center for Public Health and Nutrition, National Research and Innovation Agency (BRIN), Bogor, Indonesia

¹²Research Center for Pharmaceutical Ingredients and Traditional Medicine, 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: 22/11/2025 Revised: 14/04/2026 Accepted: 27/04/2026 Published: 31/05/2026

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Abstract

Background: Escherichia coli (E. coli) is a common commensal bacterium in poultry and a key indicator of antimicrobial resistance (AMR). The emergence of extended-spectrum β-lactamase (ESBL)-producing strains, particularly those carrying cefotaximase (CTX-M) genes, poses a significant One Health concern. However, data on CTX-M–producing E. coli in quails from traditional markets in Surabaya are limited.

Aim: This study aimed to detect the CTX-M gene in E. coli isolates obtained from quail cloacal swabs from traditional markets in Surabaya and to assess their antimicrobial resistance profiles.

Methods: A total of 100 cloacal swab samples were collected from quails in five traditional markets from November to December 2024. Escherichia coli isolation and identification were performed using Eosin Methylene Blue Agar, Gram staining, and biochemical tests. The Kirby-Bauer disk diffusion method was also used to assess antibiotic sensitivity in accordance with the Clinical and Laboratory Standards Institute guidelines. The CTX-M gene was then detected in aztreonam (ATM)-resistant isolates using polymerase chain reaction (PCR).

Results: Of the 100 samples, 98% were confirmed to be E. coli. High resistance to ciprofloxacin (50%), tetracycline (33.67%), and ATM (20.4%) was observed. Four isolates (4.08%) demonstrated multidrug resistance. Among 20 ATM-resistant isolates, eight tested positive for CTX-M genes by PCR. Resistance patterns differed among markets due to differences in antimicrobial usage or exposure.

Conclusion: The high prevalence of E. coli and the detection of CTX-M–positive isolates indicate that traditional markets may act as reservoirs of ESBL-producing bacteria in quails. These findings underscore the need for strengthened AMR surveillance, improved hygiene practices, and One Health-oriented interventions along the quail production and distribution chain.

Keywords: E. coli, ESBL, MDR, Quail, Public health.

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Introduction

Escherichia coli is a commensal bacterium found in the digestive tract of poultry, including quail (Coturnix coturnix japonica), a species increasingly farmed and consumed in Indonesia, particularly in Surabaya (Rahayu et al., 2025). Although most E. coli strains are non-pathogenic, they can acquire virulence factors and antimicrobial resistance (AMR) genes, making them useful indicators for monitoring resistance dynamics in livestock (Farizqi et al., 2023; Faridah et al., 2023). Resistance to β-lactam antibiotics has become a global concern due to their widespread use in poultry for therapy, prophylaxis, and production enhancement (Widodo et al., 2023a, 2024). This use can select for strains producing extended-spectrum β-lactamases (ESBLs) that hydrolyze advanced β-lactams, limiting treatment options (Putra et al., 2019; Ahadini et al., 2025).

Among the ESBLs, cefotaxime cefotaximase (CTX-M) is the most prevalent in poultry in East Java, including Surabaya, as it efficiently degrades cefotaxime and spreads it between bacterial populations via plasmids (Adamski et al., 2015; Putri et al., 2024; Salinas et al., 2024). Its presence in quail highlights their potential role as a reservoir of resistant E. coli, with implications for animal, environmental, and human health, aligning with the One Health framework (Zhao and Hu, 2013; Rana et al., 2022).

In recent years, quail production and consumption in Indonesia, particularly in Surabaya, have intensified, with traditional markets serving as major distribution points (Oke et al., 2025). These markets often have variable hygiene practices and handle multiple animal species, which may facilitate the accumulation and transmission of resistant bacteria (Wardhana et al., 2021; Martinez-Laorden et al., 2023). Cloacal swabs are considered representative samples for detecting ESBL-producing E. coli because they contain both gut microbiota and waste excretions (Williams and Athrey, 2020).

However, data on CTX-M-carrying E. coli in quail from Surabaya markets are limited, creating a clear research gap in East Java regarding the prevalence and distribution of ESBL-producing E. coli in poultry. Most previous studies in Indonesia have focused on chickens and broilers, with few examining quail or traditional market settings, limiting comprehensive AMR surveillance and mitigation strategies (Tyasningsih et al., 2025).

Molecular detection using polymerase chain reaction (PCR) allows accurate identification of ESBL genes, providing insight into the distribution of AMR in poultry food sources and supporting evidence-based interventions (Rahayu et al., 2025; Tayh et al., 2025). In this study, we aimed to detect the CTX-M gene in E. coli from cloacal swabs of quail collected from traditional markets in Surabaya. The study is expected to provide local data to strengthen AMR databases, inform One Health strategies, and guide policymakers, veterinary authorities, and producers in improving poultry health management and food safety.

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Materials and Methods

Research design

The study was conducted from November to December 2024 and involved 100 cloacal swab samples of quail collected from five traditional markets in Surabaya: Turi, Bratang, Cemara Pabean, Kupang, and Benowo, with 20 samples from each market. Sampling was performed using a random sampling method: 20 quails were selected from different vendors in each market, and individual quails were chosen at regular intervals within each vendor to ensure unbiased representation. This laboratory-based exploratory and observational study was conducted at the Veterinary Public Health Laboratory, Faculty of Veterinary Medicine, Wijaya Kusuma University, Surabaya.

The culture media used included Eosin Methylene Blue Agar (EMBA; Oxoid®, CM0069, UK), Triple Sugar Iron Agar (TSIA; HiMedia®, India, M021), Simmons Citrate Agar (SCA; HiMedia®, India, M099), Sulfide Indole Motility (SIM; HiMedia®, India, M181), Methyl Red (MR; HiMedia®, India, GM070), Voges–Proskauer (VP; HiMedia®, India, GM070), and Mueller–Hinton Agar (HiMedia®, India, M173).

Isolation and identification of E. coli strains

Each cloacal swab sample was first inoculated into buffered peptone water for enrichment and then grown on EMBA (HiMedia®, India, CM0069), a selective and differential medium containing peptone, lactose, sucrose, eosin, and methylene blue. Methylene blue inhibits Gram-positive bacteria, allowing selective isolation of Gram-negative enteric bacteria, while eosin and methylene blue act as pH indicators. Lactose and sucrose serve as carbohydrate sources for coliform differentiation (Ariyanti et al., 2025).

Colonies displaying a metallic green sheen on EMBA were considered to be presumptive E. coli. These colonies were further examined by Gram staining and confirmed using a series of biochemical tests, including (Triple sugar iron Agar (TSIA); HiMedia®, India, M021), (SCA; HiMedia®, India, M099), (SIM; HiMedia®, India, M181), and (MR-VP; HiMedia®, India, GM070) (Mustika et al., 2024).

Phenotypic methods (culture characteristics, biochemical tests, and antibiotic susceptibility profiling) were combined with genotypic confirmation using PCR detection of the CTX-M gene in aztreonam (ATM)-resistant isolates to strengthen identification validity. This integrated approach ensures the accurate identification of ESBL-producing E. coli in quail samples.

Antibiotic resistance testing

Antibiotic susceptibility testing was performed using the Kirby–Bauer disc diffusion method according to the Clinical and Laboratory Standards Institute guidelines (CLSI, 2020). The following antibiotics were tested: (ATM, 30 µg), ciprofloxacin (CIP, 5 µg), tetracycline (TE, 30 µg), kanamycin (K, 30 µg), and chloramphenicol (C, 30 µg).

The inhibition zone diameter was measured, and isolates were categorized as resistant, intermediate, or sensitive according to the CLSI standards. In this study, intermediate isolates were grouped with resistant isolates to provide a conservative estimate of resistance prevalence. Isolates exhibiting resistance to three or more antibiotic classes were defined as multidrug resistance (MDR) (Ahadini et al., 2025).

DNA extraction

Genomic DNA was extracted from ATM-resistant E. coli isolates using the Qiagen® DNeasy Blood and Tissue Kit (Germany) according to the manufacturer’s instructions. The quality and concentration of the extracted DNA were measured using a spectrophotometer to ensure suitability for PCR.

Polymerase chain reaction

Molecular detection of the CTX-M gene in E. coli isolates was performed using PCR. PCR reaction mixtures contained 12.5 µl 2× PCR master mix, 1 µl of each primer (forward and reverse, 10 µM), 5 µl DNA template, and nuclease-free water to a final volume of 25 µl. PCR amplifies specific DNA fragments through repeated denaturation, primer annealing, and extension cycles using the heat-stable Taq DNA polymerase enzyme (Laksmi et al., 2025).

The primers used were as follows:

Forward: 5’–ATGTGCAGYACCAGTAARGT–3’

Reverse: 5’–TGGGTRAARTARCTSACCAGA–3’

These primers target a single 593-bp fragment of the CTX-M gene, not two pairs as previously stated, detecting major CTX-M groups, including CTX-M-1, CTX-M-2, and CTX-M-9.

The PCR cycling conditions were as follows: initial denaturation at 95°C for 5 minutes; 35 cycles of denaturation at 95°C for 30 seconds, annealing at 57°C for 30 seconds, and extension at 72°C for 45 seconds; and a final extension at 72°C for 7 minutes. Positive controls included E. coli isolates previously confirmed to carry the CTX-M gene, whereas negative controls included sterile nuclease-free water. PCR products were analyzed using 1.5% agarose gel electrophoresis, and the presence of a 593-bp band indicated CTX-M gene detection (Ferreira et al., 2011).

Data analysis

All data were compiled and analyzed descriptively using SPSS version 28. The prevalence of E. coli and patterns of antibiotic resistance were summarized for each market using percentages and frequency distributions. No inferential statistical comparisons were performed; differences between markets were presented descriptively to provide an overview of variation across the five traditional markets.

Ethical approval

Ethical approval for this study was obtained from the Animal Ethics Committee, Faculty of Veterinary Medicine, Wijaya Kusuma University Surabaya, Indonesia, with approval number 170-KKE-2025, issued on January 23, 2025.

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Results

Of 100 quail cloacal swab samples obtained from five traditional markets in Surabaya, 98 (98%) were successfully identified as E. coli. The Turi, Bratang, and Cemara Pabean markets showed the highest isolation rate (100%), whereas the Kupang and Benowo markets showed slightly lower levels (95%). These findings indicate a high level of E. coli contamination in the quail sales chain at traditional markets (Table 1). Colonies grown on EMBA media appeared metallic green, a characteristic of E. coli due to lactose fermentation (Fig. 1). Gram staining revealed a pink bacillus morphology, indicating that the isolate was gram-negative (Fig. 2).

Fig. 1. (A) Growth of E. coli colonies on EMBA agar showing a characteristic metallic green sheen. (B) Growth of E. coli colonies on MCA agar showing pink lactose-fermenting colonies.

Fig. 2. Microscopic image of E. coli isolates observed under a light microscope at 1,000× magnification.

Biochemical test results supported this identification, indicated by a color change from red to yellow in all TSIA tubes, indicating the ability to ferment glucose, lactose, and sucrose. Positive gas production was detected, while H₂S formation was negative. The SCA test yielded a negative result, whereas the SIM test showed positive indole formation after the addition of Kovac’s reagent. The MR test produced a positive red color change, and the VP test showed no negative color change (Fig. 3). This combination of results confirms the typical characteristics of E. coli.

Fig. 3. The biochemical test of E. coli isolates, including TSIA, SCA, SIM, and MR-VP tests.

Antibiotic resistance testing of 98 E. coli isolates showed the highest resistance to CIP (50%), followed by TE (33.67%) and ATM (20.4%). In contrast, kanamycin (9.18%) and chloramphenicol (7.14%) resistance was relatively low (Table 2). Differences between markets were quite clear; for example, Kupang Market had ATM resistance (5.26%) and CIP resistance (84.21%), while Bratang Market showed a lower level.

Of the total isolates, four (4.08%) exhibited MDR, indicating resistance to at least three antibiotic groups. Cemara Pabean Market had the highest MDR percentage (10%), followed by Turi (5%) and Kupang (5.26%), while no MDR isolates were found in Bratang and Benowo (Table 3). Three isolates (PTU29, PKU21, and PCP25) exhibited resistance to the three antibiotic groups, with a pattern dominated by a combination of ATM and CIP. One isolate (PCP26) exhibited the broadest resistance pattern, resisting all tested antibiotics (TE, C, ATM, K, and CIP) (Table 4). Figure 4 shows the E. coli isolates exhibiting this MDR pattern.

Isolates resistant to ATM were further analyzed using PCR to detect the CTX-M gene. Of the 20 ATM resistant E. coli isolates, eight tested positive for CTX-M (Figs. 5 and 6).

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Discussion

The results of this study indicate a very high level of E. coli contamination in quail sold in traditional markets in Surabaya, with 98% of samples testing positive. This finding is consistent with previous reports showing that small birds, including quail, can serve as important reservoirs of enteric bacteria (Martinez-Laorden et al., 2023). High contamination levels are likely related to environmental factors in rearing, inconsistent sanitation, and unhygienic handling practices during distribution and sales (Agustin et al., 2025; Sparaciari et al., 2025). Differences in the frequency of isolations between markets—with higher rates in Turi, Bratang, and Cemara Pabean Markets and slightly lower in Kupang and Benowo—reflect variations in environmental and management practices that influence bacterial contamination. This situation confirms that traditional markets play a critical role in the circulation of commensal and pathogenic bacteria, including those potentially resistant to antibiotics (Prayudi et al., 2023; Hasan et al., 2025).

The identification of bacteria through morphological observation and biochemical tests showed consistent characteristics of E. coli, such as fermentation of three types of carbohydrates in TSIA media, positive indole results, and a positive MR and negative VP pattern. These results confirm the effectiveness of EMBA selective media combined with biochemical tests in validating enteric isolates from cloacal samples of quail (Mustika et al., 2024).

Antibiotic resistance profiles provide important insights into antimicrobial selection patterns in commensal E. coli populations in Surabaya, Indonesia. High resistance to CIP (50%), TE (33.67%), and ATM (20.4%) indicates strong selection pressure, likely due to routine use in poultry farming. However, this study did not directly measure antibiotic usage at the farm level; therefore, the association between observed resistance patterns and on-farm antibiotic practices is inferred from existing literature and regional reports rather than direct usage data (Smith et al., 2023). Dominant resistance patterns—particularly to CIP, TE, and ATM—highlight antimicrobial exposure along the poultry distribution chain. CIP and tetracycline are commonly used for treatment and prophylaxis in poultry; however, high resistance may reflect overuse or lack of regulation (Ali et al., 2025). Resistance to ATM, although lower, is concerning because it belongs to the β-lactam class, rarely recommended for production animals (Varela et al., 2021).

Variations in resistance patterns across markets further highlight differences in management practices and antibiotic exposure at quail supply sources (Rahayu et al., 2025). Nevertheless, these differences should be interpreted cautiously without direct data on antibiotic administration, biosecurity practices, and farm management from each supplier. These findings reinforce the notion that practices along the supply chain—from farms to markets—may significantly influence AMR dynamics (Elbehiry and Marzouk, 2025).

The presence of four MDR isolates (4.08%) indicates that quail can act as reservoirs of resistant bacteria with the potential to spread to the environment, contaminate food, and affect humans. Notably, PCP26 was resistant to all tested antibiotics, indicating that it carries a plasmid with high horizontal transfer potential (Coluzzi and Rocha, 2025). Resistance to ATM and CIP was the most common MDR combination, reflecting selective exposure to these antibiotics in farm environments (Pereira et al., 2024).

The detection of the CTX-M gene in eight ATM-resistant isolates is significant for understanding potential plasmid-mediated resistance transfer (Widodo et al., 2023b). The CTX-M gene facilitates the formation of ESBL, and its presence in commensal poultry bacteria increases the risk of horizontal gene transfer to pathogenic bacteria or human-associated bacteria via contact, contaminated environments, or food consumption (Khairullah et al., 2024). CTX-M-1, CTX-M-2, and CTX-M-9 groups are commonly reported in poultry isolates in Southeast Asia, with CTX-M-15 frequently identified in Indonesia and neighboring countries (Faridah et al., 2023). The positivity rate observed in this study (8/20 ATM-resistant isolates) is comparable to reports from other regions in Indonesia and several Southeast Asian countries, where the prevalence of CTX-M in poultry-associated E. coli ranges from low to moderate levels depending on the sampling source and detection methods. However, this study did not include sequencing analysis; therefore, specific CTX-M variants could not be determined (Ayinla and Mateus, 2023).

Additionally, the study was conducted in a single city (Surabaya); therefore, the findings may not represent conditions in other regions. Other limitations include the relatively limited sample size, the focus on traditional markets only (excluding farms and slaughterhouses), and the absence of molecular typing methods, such as sequencing or plasmid characterization, to explore genetic relatedness and transmission dynamics (Khairullah et al., 2025). Variations in market hygiene, antibiotic use without veterinary oversight, and suboptimal waste management can exacerbate the spread of AMR in dense urban areas (Manyi-Loh et al., 2018; Putri et al., 2024).

This study confirms that the supply and distribution chain of small poultry, including quail, contributes to the dynamics of AMR and may serve as a pathway for the spread of ESBL-producing E. coli. Effective AMR control requires integrated strategies, including routine monitoring of production animals, stricter antibiotic regulation, improved market hygiene, and farmer/trader education (Ansharieta et al., 2021; Trinchera et al., 2025). Integrating livestock resistance data into national surveillance systems will strengthen mitigation efforts and support Indonesia’s response to the AMR threat (Coyne et al., 2019).

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Conclusion

This study shows high levels of E. coli contamination in quail traded in traditional markets in Surabaya, with a prevalence of 98%. Antibiotic resistance analysis revealed alarming resistance patterns, particularly to CIP, TE, and ATM, and MDR isolates also identified. The CTX-M gene was detected in eight ATM-resistant E. coli isolates, confirming the potential for ESBL spread through small poultry populations. These findings highlight the important role of quail as a reservoir of AMR and emphasize the need for ongoing surveillance, more judicious use of antibiotics, and the application of One Health principles to control the spread of AMR in the food and environmental sectors.

Further studies are recommended to include larger sample sizes across multiple regions, direct assessment of antibiotic usage at the farm level, and molecular characterization, such as sequencing of CTX-M variants and plasmid analysis, to better understand transmission dynamics and genetic relatedness of resistant isolates.

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Acknowledgments

This study was supported by the Faculty Research Group Program of the Faculty of Veterinary Medicine, Universitas Airlangga, under the 2025 Research Contract No. 2989/B/UN3.FKH/PT.01.03/2025.

Conflict of interest

The authors declare no conflict of interest.

Funding

This study was supported by the Faculty Research Group Program of the Faculty of Veterinary Medicine, Universitas Airlangga, under the 2025 Research Contract No. 2989/B/UN3.FKH/PT.01.03/2025.

Author’s contributions

W.T., M.O.K., U.R., and F.J.W. Conceptualization and design of the study. M.H.E., W.W., and I.A.K.: Data acquisition. A.R.K., S.R., and I.F.M.: data analysis and interpretation. B.P.P., D.A.A.K., and J.Y.H.T.: Preparing the original draft. M.F.R.P. and R.Z.A.: Writing, reviewing, and editing. All authors have read and approved the published version of the manuscript.

Data availability

All data are available in the revised manuscript.

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Table 1. Prevalence of E. coli in cloacal swabs from quail from different regions.

Table 2. Antibiotic susceptibility of E. coli isolates from different regions of the world.

Note: ATM, ciprofloxacin, TE, kanamycin, chloramphenicol, resistant.

Fig. 4. Antibiotic sensitivity test of the E. coli isolates Numbers 1–5 indicate the antibiotics tested: (1) ATM; (2) TE; (3) CIP; (4) kanamycin; and (5) chloramphenicol.

M K K+ PTU25 PTU28 PTU29 PKU21 PCP23 PCP25 PCP26 PTU5 PTU24

Table 3. Results of MDR tests on E. coli isolates from different regions.

Table 4. Resistance patterns of MDR-resistant E. coli isolates.

Note: Tetracycline (TE), chloramphenicol (C), azoteonam (ATM), kanamycin (K), ciprofloxacin (CIP), resistant (R), sensitive (S).

Fig. 5. PCR amplification products of ATM-resistant E. coli isolates for the CTX-M gene at 593 bp. Lane labels: M=DNA marker; K–=negative control; K+=positive control; PTU25, PTU28, PTU29, PKU21, PCP23, PCP25, PCP26, PTU5, and PTU24=sample isolates.

M PTU29 PKU8 PKU22 PBR9 PBR22 PBR23 PBR28
PBR29 PBR30 PBW1 PCP13

Fig. 6. PCR amplification products of ATM-resistant E. coli isolates for the CTX-M gene at 593 bp. Lane labels: M=DNA marker; K–=negative control; K+=positive control; PTU29, PKU8, PKU22, PBR9, PBR22, PBR23, PBR28, PBR29, PBR30, PBW1, and PCP13=sample isolates.