| Summary: | The emergence and dissemination of <i>Escherichia coli</i> (<i>E. coli</i>) 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 <i>Escherichia coli</i> isolates from chickens in central China during 2016–2019. A total of 407 <i>E. coli</i> 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 <i>E. coli</i> 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 <i>bla</i><sub>TEM</sub> + <i>bla</i><sub>CTX-M</sub> (74.46%), in which the frequency of carriers increased steadily, followed by <i>bla</i><sub>CTX-M</sub> + <i>bla</i><sub>SHV</sub> (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 <i>bla</i><sub>CTX-M</sub>, <i>bla</i><sub>TEM</sub>, and <i>bla</i><sub>SHV</sub>, with limits of detection of 2.06 × 10<sup>1</sup> copies/μL, 1.10 × 10<sup>1</sup> copies/μL, and 1.86 × 10<sup>1</sup> 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 <i>bla</i><sub>CTX-M</sub>, <i>bla</i><sub>TEM</sub>, and <i>bla</i><sub>SHV</sub>, respectively. In conclusion, our findings indicated that ESBL-producing <i>E.coli</i> 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.
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