| Summary: | Chicken products and chickens with colibacillosis are often reported to be a suspected source of extraintestinal pathogenic <i>Escherichia coli</i> (ExPEC) causing several diseases in humans. Such pathogens in healthy chickens can also contaminate chicken carcasses at the slaughter and then are transmitted to humans via food supply; however, reports about the ExPEC in healthy chickens are still rare. In this study, we determined the prevalence and characteristics of ExPEC isolates in healthy chickens in China. A total of 926 <i>E. coli</i> isolates from seven layer farms (371 isolates), one white-feather broiler farm (78 isolates) and 17 live poultry markets (477 isolates from yellow-feather broilers) in 10 cities in China, were isolated and analyzed for antibiotic resistance phenotypes and genotypes. The molecular detection of ExPEC among these healthy chicken <i>E. coli</i> isolates was performed by PCRs, and the serogroups and antibiotic resistance characteristics of ExPEC were also analyzed. Pulsed-field gel electrophoresis (PFGE) and Multilocus sequence typing (MLST) were used to analyze the genetic relatedness of these ExPEC isolates. We found that the resistance rate for each of the 15 antimicrobials tested among <i>E. coli</i> from white-feather broilers was significantly higher than that from brown-egg layers and that from yellow-feather broilers in live poultry markets (<i>p</i> < 0.05). A total of 22 of the 926 <i>E. coli</i> isolates (2.4%) from healthy chickens were qualified as ExPEC, and the detection rate (7.7%, 6/78) of ExPEC among white-feather broilers was significantly higher than that (1.6%, 6/371) from brown-egg layers and that (2.1%, 10/477) from yellow-feather broilers (<i>p</i> < 0.05). PFGE and MLST analysis indicated that clonal dissemination of these ExPEC isolates was unlikely. Serogroup O78 was the most predominant type among the six serogroups identified in this study, and all the six serogroups had been frequently reported in human ExPEC isolates in many countries. All the 22 ExPEC isolates were multidrug-resistant (MDR) and the resistance rates to ampicillin (100%) and sulfamethoxazole-trimethoprim (100%) were the highest, followed by tetracycline (95.5%) and doxycycline (90.9%). <i>bla</i><sub>CTX-M</sub> was found in 15 of the 22 ExPEC isolates including 10 harboring additional fosfomycin resistance gene <i>fosA3</i>. Notably, plasmid-borne colistin resistance gene <i>mcr-1</i> was identified in six ExPEC isolates in this study. Worryingly, two ExPEC isolates were found to carry both <i>mcr-1</i> and <i>bla</i><sub>NDM</sub>, compromising both the efficacies of carbapenems and colistin. The presence of ExPEC isolates in healthy chickens, especially those carrying <i>mcr-1</i> and/or <i>bla</i><sub>NDM</sub>, is alarming and will pose a threat to the health of consumers. To our knowledge, this is the first report of <i>mcr-1</i>-positive ExPEC isolates harboring <i>bla</i><sub>NDM</sub> from healthy chickens.
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