Prevalence of Extended-Spectrum β-Lactamase-Resistant Genes in Escherichia coli Isolates from Central China during 2016–2019

Simple Summary β-lactam antibiotics are commonly used for the treatment of severe infection in both animals and humans, and the resistance of E. coli to third-generation cephalosporins is becoming a worldwide problem. Our results revealed 407 E. coli strains isolated from central China exhibited strong resistance to first- to fourth-generation cephalosporins and monobactam antibiotics, and piperacillin/tazobactam was the most effective drug. Phenotypically, 63.88% of the isolates were positive for ESBL production and the isolation rates kept growing. Genetic characterization identified CTX-M as the most prevalent type and blaTEM + blaCTX-M as the most common ESBL genotype combination. Furthermore, a novel multiplex real-time PCR method for detecting the three most common ESBL genes was developed as a complementary rapid-screening test for antimicrobial resistance genes. These results confirm the cephalosporins resistance urgency in the central Chinese poultry sector and suggest the continuous monitoring and timely detection of ESBL-producing isolates is vitally important in establishing appropriate antimicrobial therapies and blocking their transmission. Abstract The emergence and dissemination of Escherichia coli (E. coli) strains that produce extended-spectrum beta-lactamases (ESBLs) represents a major public health threat. The present study was designed to evaluate the prevalence and characteristics of ESBL-producing Escherichia coli isolates from chickens in central China during 2016–2019. A total of 407 E. coli strains isolated from 581 chicken swabs were identified conventionally and analyzed for various cephalosporin susceptibility by disk-diffusion assay. ESBL-producing strains were screened using the double=disk synergy test and ESBL-encoding genes were carried out by PCR/sequencing. A total of 402 E. coli isolates exhibited strong resistance to first- to fourth-generation cephalosporins and monobactam antibiotics, especially cefazolin (60.69%), cefuroxime (54.05%), cefepime (35.14%), ceftriaxone (54.30%), and aztreonam (40.29%). Piperacillin/tazobactam (1.72%) was the most effective drug against the strains, but the resistance rates increased each year. Among the isolates, 262 were identified as ESBL producers and the isolation rates for the ESBL producers increased from 63.37% to 67.35% over the four years. CTX-M (97.33%) was the most prevalent type, followed by TEM (76.72%) and SHV (3.05%). The most common ESBL genotype combination was blaTEM + blaCTX-M (74.46%), in which the frequency of carriers increased steadily, followed by blaCTX-M + blaSHV (3.05%). In addition, the most predominant specific CTX-M subtypes were CTX-M-55 (48.47%) and CTX-M-1 (17.94%), followed by CTX-M-14 (11.01%), CTX-M-15 (8.02%), CTX-M-9 (6.11%), CTX-M-65 (4.58%), and CTX-M-3 (1.15%). Moreover, a novel multiplex qPCR assay was developed to detect blaCTX-M, blaTEM, and blaSHV, with limits of detection of 2.06 × 101 copies/μL, 1.10 × 101 copies/μL, and 1.86 × 101 copies/μL, respectively, and no cross-reactivity with other ESBL genes and avian pathogens. The assays exhibited 100% sensitivity and specificities of 85%, 100%, and 100% for blaCTX-M, blaTEM, and blaSHV, respectively. In conclusion, our findings indicated that ESBL-producing E.coli strains isolated from chickens in central China were highly resistant to cephalosporins and frequently harbored diversity in ESBL-encoding genes. These isolates can pose a significant public health risk. The novel multiplex qPCR method developed in this study may be a useful tool for molecular epidemiology and surveillance studies of ESBL genes.


Introduction
Escherichia coli (E. coli) is one of the main pathogens that cause diarrhea in pigs and chickens, and it is an important cause of death among piglets and young poultry [1]. Antibiotics are generally used to treat E. coli infections due to the lack of an effective vaccine. However, bacteria with resistance to multiple drugs have increasingly been found because of the overuse and misuse of antibiotics on farms to reduce and prevent the risk of infection [2]. Pigs and chickens are food-producing animals but are also major reservoirs of organisms with antimicrobial resistance [3,4]. In addition to antibiotic residues in food, antibiotic resistance genes can disseminate between animals and humans via the food chain [5]. Therefore, drug-resistant E. coli strains pose severe hazards to animal husbandry and food safety.
Resistance to third-generation cephalosporins is growing, so much attention has been increasingly focused on extended-spectrum β-lactamase (ESBL) enzymes that can hydrolyze the β-lactam ring in a wide range of β-lactam antimicrobials [6]. ESBL-producing E. coli isolates are also among the most critical antibiotic-resistant pathogens categorized by the World Health Organization. In general, the most prevalent ESBLs are encoded by bla TEM , bla CTX-M , and bla SHV , and the frequency of carrying bla CTX-M is highest in ESBLproducing E. coli. Up to 97.8% of E. coli isolates from pigs, cattle, chicken, and sheep produce CTX-M group enzymes [7]. In recent years, the extensive use of antimicrobial agents, coupled with the transmissibility of resistance determinants, such as integrons, has resulted in the emergence and spread of MDR ESBL-producing strains. Previous studies have demonstrated that E. coli is one of the major causes of childhood diarrhea, and the distribution of class 1 integrons and ESBLs in the strains is highly prevalent [8]. The production of ESBLs can greatly restrict the uses of clinical medication, so the continuous monitoring and timely detection of ESBL-producing isolates is vitally important in establishing appropriate antimicrobial therapies and blocking their transmission [9]. At present, the common methods used to identify ESBL-producing bacteria include the drug-sensitive disk-diffusion method, microbroth-dilution method, and E-test method. However, these are culture-based methods for phenotypic screening, which are time-consuming, laborious, and incapable of distinguishing different ESBL genotypes. Molecular techniques, such as multiplex PCR and real-time PCR, have been applied to detect the specific genes that encode ESBLs [10,11]. The emergence of ESBL enzymes is mainly due to plasmids and chromosomal mutations [12]. Unfortunately, many new mutations have been found because of the misuse of third-generation cephalosporins, thereby leading to the emergence of new drug-resistant gene subtypes. For example, the CTX-M genotypes include CTX-M-1, CTX-M-2, CTX-M-8, CTX-M-9, and CTX-M-25 [13]. Therefore, it is necessary to improve the existing molecular detection techniques. In the present study, we developed a novel multiplex real-time PCR method to detect the three most common ESBL genes (bla TEM , bla CTX-M , and bla SHV ) and to identify the trends in cephalosporin resistance among E. coli isolates obtained from chicken feces samples collected in central China during 2016-2019.

Collection of Bacterial Strains
From 2016 to 2019, a total of 581 swab samples were obtained from broiler chickens at different farms located in central China, including Wuhan, Xiangyang, Xiantao, Xinyang, Daye, Tianmen, Jinan, Hanchuan, Changde, and Yucheng. The collected samples were Animals 2022, 12, 3191 3 of 12 streaked onto MacConkey agar and eosin-methylene blue agar (Haibo, Qingdao, China), and then incubated at 37 • C for 24 h. Typical E. coli colonies were subsequently confirmed by PCR amplification of the phoA gene using E. coli-specific primers. In addition, reference strain ATCC 29522 was obtained from the China Veterinary Culture Collection Center. Unless otherwise stated, all cultures were maintained in Luria broth (Haibo) in the presence of oxygen at 37 • C.

Antimicrobial Susceptibility Testing
The susceptibility of E. coli isolates to antimicrobial agents was determined using the disk-diffusion assay according to the methods of the Clinical and Laboratory Standards Institute (CLSI). Briefly, antimicrobial disks were placed on the surfaces of Mueller-Hinton agar (Haibo) plates inoculated with E. coli. The inoculated plates were incubated at 37 • C for 24 h, before the diameters of the zones of inhibition were measured and interpreted following CLSI guidelines. The E. coli ATCC 25922 strain was used for quality control in the test. The antimicrobial disks (Oxoid, England, UK) contained cefazolin (CZO), cefuroxime (CXM), cefoxitin (FOX), ceftriaxone (CRO), ceftazidime (CAZ), cefoperazone/sulbactam (SCF), cefepime (FEP), aztreonam (ATM), amoxicillin/clavulanic acid (AMC), ampicillin/sulbactam (SAM), and piperacillin/tazobactam (TZP).

Screening for ESBL-Producing Strains
The E. coli isolates were phenotypically screened using the double-disk synergy test. Briefly, antimicrobial disks containing CAZ (30 µg) and CAZ/clavulanic acid (30/10 µg), or cefotaxime (30 µg) and cefotaxime/clavulanic acid (30/10 µg) (Solarbio, Beijing, China), were placed on the surfaces of Mueller-Hinton agar plates inoculated with E. coli. After inverted culture at 37 • C for 24 h, the diameters of the zones of inhibition were measured. ESBL production was indicated when the zone of inhibition in the CAZ or cefotaxime group was expanded by clavulanate (≥5 mm). E. coli ATCC 25922 was used as the negative control.

Characterization of ESBL Genes by PCR Sequencing
The ESBL genes (bla TEM , bla CTX-M , and bla SHV ) were detected in ESBL-producing E. coli isolated by PCR and confirmed by DNA sequencing. Briefly, nucleic acid was extracted from the isolates using the boiling method. PCR was performed using gene-specific primers (Table 1) and the products were sequenced by Sangon Biotech (Shanghai, China).

Probe and Primer Design
Genetic sequences of bla TEM , bla CTX-M , and bla SHV were downloaded from the Gen-Bank database and aligned using Megalign (Madison, WI, USA). The primers and probes were designed using Primer Express 3.0 (ABI, La Jolla, CA, USA) software based on the conserved sequences (Table 1). Subsequently, the specificities of the primers and probes were evaluated by BLAST searching against the GenBank non-redundant database. Appropriate fluorophore groups and quenched groups were labeled at the 5 and 3 ends of the probes ( Table 1). All primers were synthesized by Sangon Biotech.

Multiplex Quantitative PCR Amplification
The optimal qPCR system contained 5 µL of AceQ ® qPCR Probe Master Mix (Vazyme, Nanjing, China), 0. The optimum reaction temperature was tested from 55.6 • C to 64.6 • C, with a temperature gradient of 1.5 • C. The specificity of the qPCR assay was examined with E. coli strains, including ESBL-producing strains and E. coli ATCC 25922. Nuclease-free H 2 O was used for the negative controls. Amplification and signal detection were conducted with a Bio-Rad system (Bio-Rad, Hercules, CA, USA).

Sensitivity Tests and Standard Curve
The detection targets were cloned into the pMD18-T vector and the concentrations of pMD-CTX-M, pMD-TEM, and pMD-SHV were determined using a NanoDrop One/One C spectrophotometer (Thermo, Waltham, MA, USA). Sensitivity and standard curve tests were conducted using a 10-fold dilution series of the recombinant plasmids. Nucleasefree H 2 O was used as the negative control. Sensitivity tests were performed under the optimized reaction conditions and the results were compared to the PCR results. Standard curves were also established.

Detection of ESBL Genes Using the Novel Multiplex qPCR Method
All of the ESBL-positive E. coli isolates were screened for the presence of bla TEM , bla CTX-M , and bla SHV using the novel multiplex qPCR method. The results obtained by multiplex qPCR were compared to those produced using the normal PCR method. The agreement between the two methods was measured based on the significance using kappa (κ) statistics in QuickCals software (https://www.graphpad.com/quickcalcs/kappa1 accessed on 5 May 2022) (Montrose, CA, USA). The sensitivity and specificity were measured with MedCalc software (https://www.medcalc.org/calc/diagnostic_test.php accessed on 5 May 2022) (Ostend, Belgium).
During 2016-2019, 407 E. coli strains were obtained using cloacae swabs chickens in central China and 64.37% (262/407) were ESBL producers (   72%). TZP was the most potent antibiotic with a susceptibility of 98.28%, follow SCF (93.86%), but the resistance rates to TZP and SCF tended to increase during the years ( Figure 1). Similar to the phenotypic test results, the resistance rates to the antibiotics were lower in 2017. In addition, all of the ESBL producers (100%, 262/262) resistant to at least one or more β-lactam antibiotic, and the resistance rate was 1 (24/145) in non-ESBL producers, which increased rapidly ( Table 2).

Genotypic Characterization of ESBL Producers
Among the 262 ESBL-positive isolates, all isolates were positive for ESBL genes, thereby indicating the high sensitivity of the phenotypic tests (Table 3). Among the ESBLpositive isolates, bla CTX-M genes were detected in 97.33% (255/262) of the isolates and bla TEM genes were detected in 76.72% (201/262) of the isolates. However, bla SHV genes had the lowest detection rate of (3.05%) 8/262, and they were only detected in 2019. In addition, the most common ESBL genotype combination in E. coli was bla TEM + bla CTX-M (194/262, 74.46%), and the rate increased steadily (Table 3). By contrast, the detection rates for bla TEM

Genotypic Characterization of ESBL Producers
Among the 262 ESBL-positive isolates, all isolates were positive for ESBL genes, thereby indicating the high sensitivity of the phenotypic tests (Table 3). Among the ESBLpositive isolates, blaCTX-M genes were detected in 97.33% (255/262) of the isolates and blaTEM genes were detected in 76.72% (201/262) of the isolates. However, blaSHV genes had the lowest detection rate of (3.05%) 8/262, and they were only detected in 2019. In addition, the most common ESBL genotype combination in E. coli was blaTEM+blaCTX-M (194/262, 74.46%), and the rate increased steadily (Table 3). By contrast, the detection rates for blaTEM

Establishment of a Novel Multiplex qPCR Method
Primers and probes were designed based on the conserved sequences in order to establish a novel multiplex qPCR method to detect the three common ESBL genes (bla TEM , bla CTX-M , and bla SHV ) (Figure 3). Some point mutations cannot be avoided when detecting the targets in the CTX-M groups, so primers and probes were inserted with degenerate bases. The optimal reaction conditions for multiplex qPCR comprised initial denaturation at 95 • C for 30 s, followed by 40 cycles at 95 • C for 5 s and 57 • C for 30 s.

Establishment of a Novel Multiplex qPCR Method
Primers and probes were designed based on the conserved sequences in ord establish a novel multiplex qPCR method to detect the three common ESBL genes (bl blaCTX-M, and blaSHV) (Figure 3). Some point mutations cannot be avoided when dete the targets in the CTX-M groups, so primers and probes were inserted with degen bases. The optimal reaction conditions for multiplex qPCR comprised initial denatura at 95 °C for 30 s, followed by 40 cycles at 95 °C for 5 s and 57 °C for 30 s. In the qPCR specificity tests, only ESBL-producing strains produced three pos fluorescence signals, and no positive signals were observed with ATCC 29522 nuclease-free H2O ( Figure 4A). In the qPCR specificity tests, only ESBL-producing strains produced three positive fluorescence signals, and no positive signals were observed with ATCC 29522 and nucleasefree H 2 O ( Figure 4A).  In the qPCR sensitivity tests, the detection limits for blaCTX-M, blaTEM, and blaSHV were determined as 2.06 × 10 1 copies/μL, 1.10 × 10 1 copies/μL, and 1.86 × 10 1 copies/μL in a singletube assay system, respectively, and the limits were 2.06 × 10 3 copies/μL, 1.10 × 10 3 copies/μL, and 1.86 × 10 4 copies/μL in the PCR sensitivity tests ( Figure 5). Thus, the sensitivity of the new method was at least 100 times that of the PCR assays. Using the standard curves established for the three ESBL genes ( Figure 4B), three linear relationships were obtained between the copy number (x-axis) and quantification cycle (Cq) value (y axis) as follows: y = -3.328x + 38.561, R 2 = 0.999 for blaTEM; y = -3.309x + 38.09, R 2 = 0.998 for blaCTX-M; and y = -3.304x + 37.928, R 2 = 0.999 for blaSHV. In the qPCR sensitivity tests, the detection limits for bla CTX-M , bla TEM , and bla SHV were determined as 2.06 × 10 1 copies/µL, 1.10 × 10 1 copies/µL, and 1.86 × 10 1 copies/µL in a single-tube assay system, respectively, and the limits were 2.06 × 10 3 copies/µL, 1.10 × 10 3 copies/µL, and 1.86 × 10 4 copies/µL in the PCR sensitivity tests ( Figure 5). Thus, the sensitivity of the new method was at least 100 times that of the PCR assays. Using the standard curves established for the three ESBL genes ( Figure 4B), three linear relationships were obtained between the copy number (x-axis) and quantification cycle (Cq) value (y axis) as follows: y = −3.328x + 38.561, R 2 = 0.999 for bla TEM ; y = −3.309x + 38.09, R 2 = 0.998 for bla CTX-M ; and y = −3.304x + 37.928, R 2 = 0.999 for bla SHV .

Comparison of the Novel Multiplex qPCR Method and PCR
To determine the efficiency of the novel multiplex qPCR method, 262 ESBLproducing E. coli isolates were analyzed by multiplex qPCR and normal PCR. The results are compared in Table 4.

Comparison of the Novel Multiplex qPCR Method and PCR
To determine the efficiency of the novel multiplex qPCR method, 262 ESBL-producing E. coli isolates were analyzed by multiplex qPCR and normal PCR. The results are compared in Table 4. Table 4. Pairwise comparison of bla TEM , bla CTX-M , and bla SHV detection using multiplex qPCR and PCR (n = 262). Among the 262 strains tested, 53 (20.23%) were positive for the presence of bla CTX-M alone and eight (3.05%) were positive for the presence of the bla CTX-M + bla SHV combination using multiplex qPCR. The same results were obtained with the PCR method, which is considered the gold standard. Six strains (6/262, 2.29%) were detected as positive for bla TEM alone by multiplex qPCR and seven strains (7/262, 2.67%) were detected as positive by PCR. The strain with a different result was detected as the combination of bla CTX-M + bla TEM using multiplex qPCR, thereby resulting in the discrepancy for one of the bla CTX-M + bla TEM samples. Both assays were negative for the detection of other β-lactamase gene patterns. Therefore, the novel multiplex qPCR method obtained almost perfect agreement with the gold standard PCR method (κ = 0.921 for bla CTX-M and κ = 1.000 for both bla TEM and bla SHV ). In addition, the specificity, sensitivity, positive predictive value (PPV), and negative predictive value (NPV) results are shown in Table 5.

Discussion
E. coli infection remains a major health concern in poultry because it can be transmitted to humans through the food chain [14]. E. coli infections severely threaten the health of humans and poultry, as well as the development of the poultry industry and the economy. The resistance of E. coli to third-generation cephalosporins is a worldwide problem [2], mainly caused by the production of ESBLs. The transfer of ESBLs between bacteria is usually mediated by plasmids [15]. More than 600 ESBLs have been reported [16], and the majority are TEM, SHV, and CTX-M-type β-lactamases. A previous study showed that poultry feces could play an important role as potential reservoirs for resistant isolates and resistance genes [17]. In the present study, 407 avian E. coli strains were isolated from fecal samples collected during 2016-2019 in central China. The isolates exhibited severe resistance to firstto fourth-generation cephalosporins and monobactam antibiotics, especially CZO (60.69%), CXM (54.05%), FEP (35.14%), CRO (54.30%), and ATM (40.29%). The resistance rates were much higher than those found in other areas of China [18][19][20]. It indicated that the E. coli recovered from chickens developed serious resistance to cephalosporins; therefore, the use of these drugs should be suspended. TZP is effective against ESBL-producing Gram-negative bacteria and it is used as a first-line drug to treat serious infections [21]. In our study, TZP was the most potent antibiotic with a susceptibility of 98.28%. Although it could be used to treat the resistant isolates, the drug resistance rates increased each year, which reminds people to use antibiotics correctly and appropriately. ESBLs can hydrolyze all types of cephalosporins and monobactams, so they are the most significant in the spread of resistant determinants worldwide, mainly in E. coli [22]. The current study identified a prevalence (64.37%, 262/407) of ESBL producers. The isolation rates of ESBL producers increased from 63.37% to 67.35% during the four years, and the rates were much higher than those found in previous similar studies of chickens in China during 2004-2007 (28.8%) [23] and 2008-2014 (41.53%) [24]. Among the ESBL-positive isolates, the dominant resistance gene was bla CTX-M (97.33%), and the most common genotype combination was bla TEM + bla CTX-M (74.46%). Compared with the OXA and SHV types, the CTX-M and TEM types have spread more widely and they are now dominant in China, possibly because cefotaxime and CRO are the most commonly used clinical drugs for the treatment of bacterial diseases caused by Enterobacterales [25,26]. In addition, the CTX-M group is used most widely in China, so CTX-M-1G (75.57%) and CTX-M-9G (21.76%) were detected in ESBL-producing strains, and the detection rate of CTX-M-9G increased each year. In some regions of Asia, CTX-M-9G is the predominant group [27] and CTX-M-9-producing E. coli are always detected in urinary tract infections [28]. Moreover, previous studies have shown that two or more CTX-M groups frequently coexist in the same isolate, which could promote the occurrence of other recombinant enzymes in the future [29]. Fortunately, CTX-M-1 and CTX-M-9 group members were not found to coexist in the isolates in the present study. Additionally, the predominant specific CTX-M types were CTX-M-55 (48.47%) and CTX-M-1 (17.94%), followed by CTX-M-14 (11.01%), CTX-M-15 (8.02%), CTX-M-9 (6.11%), CTX-M-65 (4.58%), and CTX-M-3 (1.15%). A high carrying rate of CTX-M-55 has been reported in E. coli previously in many countries, including Hong Kong and Vietnam [30,31]. The emergence of CTX-M-55 has also been detected in Salmonella strains from raw meat and food animals in China [32]. Moreover, many avian ESBL E. coli isolates can produce CTX-M-14, CTX-M-15, and CTX-M-65 commonly detected in clinical samples, which is worrying in terms of food safety. CTX-M-15 has become the most prevalent CTX-M variant in both hospitals and the environment worldwide [33]. CTX-M-3 is frequently found in Salmonella and rarely detected in E. coli. However, it should be noted that the rate of CTX-M-14, CTX-M-15, CTX-M-65, and CTX-M-3 detection increased each year in the present study. On the other hand, the phenotypic resistance to cephalosporins was not validated completely by the genotypic ESBL results, possibly due to the presence on another resistance mechanism, such as AmpC β-lactamases [34,35], which was one of the reasons for the reduction in sensitivity towards newer generations of antibiotics [36]. In addition, ESBL-producing strains with AmpC β-lactamases could cause a false-negative ESBL production [8]. In summary, our results showed that the isolates exhibited very high resistance to cephalosporins and the frequency of ESBL producers increased during 2016-2019. Therefore, the early detection and characterization of ESBLs are vitally important in establishing appropriate antimicrobial therapies and preventing their transmission.
The routine diagnostic methods currently used to detect ESBL producers involve culture-based phenotypic screening, which is time-consuming and laborious, and they cannot distinguish different ESBL genotypes [37]. Molecular detection techniques have potential advantages due to the limitations of phenotypic detection methods. In the present study, we developed a novel multiplex qPCR assay for the rapid detection of bla CTX-M , bla TEM , and bla SHV with high specificity and low detection limits. CTX-M genotypes comprise CTX-M-1, CTX-M-2, CTX-M-8, CTX-M-9, and CTX-M-25 with a low homology and a few conserved regions among each group. Therefore, it is difficult to design probes and primers to detect all of these genotypes at the same time. CTX-M-1 and CTX-M-9 were the predominant types found in avian E. coli isolates in China and we only detected these genotypes in the 262 ESBL producers by PCR in the present study. Thus, the primers used to detect CTX-M could detect CTX-M-1 and CTX-M-9 at the same time. Some point mutations cannot be avoided when detecting targets in CTX-M groups, so the primers and probe were inserted with degenerate bases. The detection limits in the multiplex qPCR assays in the single-tube assay system were determined as 2.06 × 10 1 copies/µL for bla CTX-M , 1.10 × 10 1 copies/µL for bla TEM , and 1.86 × 10 1 copies/µL for bla SHV , and they were around 100 to 1000 times higher than those using the conventional PCR method. In addition, our assays were conducted using various resistance genes and ATCC 29522. Only ESBL-producing strains produced three positive fluorescence signals, and no positive signals were obtained with ATCC 29522 and nuclease-free H 2 O. In the efficiency tests, 262 ESBLs producers were detected using the normal PCR method and multiplex qPCR assay. The qPCR assay results were in almost perfect agreement with the PCR results, except for one sample. Therefore, the results demonstrated that the novel multiplex qPCR assay was highly efficient at detection with high specificity; thus, it can be used as a clinical detection method or as a complementary rapid-screening test for antimicrobial-resistant genes while phenotypic tests are being conducted.

Conclusions
In the present study, ESBL-producing E. coli isolates from chickens in central China during 2016-2019 exhibited high resistance to cephalosporins and they frequently carried diverse ESBL genes, thereby indicating that chickens have become reservoirs of antimicrobial-resistant organisms and that the continuous monitoring and timely detection of ESBL-producing isolates is vitally important. The novel multiplex qPCR method developed in this study could be effective in monitoring the prevalence of ESBL-producing E. coli in chickens to reduce the risk of transmission to humans.