| Summary: | Antimicrobial-resistant <i>Escherichia coli</i> isolates have emerged in various ecologic compartments and evolved to spread globally. We sought to (1.) investigate the occurrence of ESBL-producing <i>E. coli</i> (ESBL-Ec) in feces from free-range chickens in a rural region and (2.) characterize the genetic background of antimicrobial resistance and the genetic relatedness of collected isolates. Ninety-five feces swabs from free-range chickens associated with two households (House 1/House 2) in a rural region in northern Tunisia were collected. Samples were screened to recover ESBL-Ec, and collected isolates were characterized for phenotype/genotype of antimicrobial resistance, integrons, and molecular typing (pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST)). Overall, 47 ESBL-Ec were identified, with the following genes detected: 35 <i>bla</i><sub>CTX-M-1,</sub> 5 <i>bla</i><sub>CTX-M-55</sub>, 5 <i>bla</i><sub>CTX-M-15</sub>, 1 <i>bla</i><sub>SHV-2,</sub> and 1 <i>bla</i><sub>SHV-12</sub>. Resistance to fluoroquinolones, tetracycline, sulfonamides, and colistin was encoded by <i>aac(6′)</i>-Ib-<i>cr</i> (<i>n</i> = 21), <i>qnr</i>B (<i>n</i> = 1), and <i>qnr</i>S (<i>n</i> = 2); <i>tet</i>A (<i>n</i> = 17)/<i>tet</i>B (<i>n</i> = 26); <i>sul</i>1 (<i>n</i> = 29)/<i>sul</i>2 (<i>n</i> = 18); and <i>mcr</i>-2 (<i>n</i> = 2) genes, respectively. PFGE and MLST identified genetic homogeneity of isolates in House 1; however, isolates from House 2 were heterogeneous. Notably, among nine identified sequence types, ST58, ST69, ST224, and ST410 belong to pandemic high-risk clonal lineages associated with extrapathogenic <i>E. coli</i>. Minor clones belonging to ST410 and ST471 were shared by chickens from both households. The virulence genes <i>fyuA, fimH, papGIII,</i> and <i>iutA</i> were detected in 35, 47, 17, and 23 isolates, respectively. Findings indicate a high occurrence of ESBL-Ec in free-range chickens and highlight the occurrence of pandemic zoonotic clones.
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