| Summary: | The increasing incidence of extended-spectrum β-lactamase (ESBL)-producing <i>Escherichia</i> (<i>E</i>.) <i>coli</i> in backyard chicken farming in Pakistan is of serious concern. This study aimed to assess the prevalence, antimicrobial resistance patterns and risk factors associated with ESBL avian pathogenic <i>E. coli</i> (APEC) isolated from backyard chickens in the Jhang district, Punjab, Pakistan. In total, 320 cloacal swabs were collected from four breeds of backyard chicken (Aseel, Golden, Misri and Necked Neck). ESBL <i>E. coli</i> were phenotypically identified using double disc synergy test (DDST) and corresponding genes were confirmed by multiplex polymerase chain reaction (mPCR). Out of the 320 samples, 164 (51.3%) were confirmed as <i>E. coli,</i> while 74 (45.1%) were characterized as ESBL <i>E. coli</i>. The frequency of isolation of ESBL <i>E. coli</i> was highest in Aseel chickens (35.1%). Of the 164 confirmed <i>E. coli</i>, 95.1%, 78.6%, 76.8%, 71.3%, 70.1%, 68.9%, 60.4% and 57.3% were resistant against tylosin, doxycycline, cefotaxime, enrofloxacin, colistin, trimethoprim/sulfamethoxazole, chloramphenicol and gentamicin, respectively. The ESBL gene types detected and their corresponding proportions were <i>bla</i><sub>CTX-M</sub> (54.1 %, 40/74), <i>bla</i><sub>TEM</sub>, (12.2%, 9/74) and co-existence (<i>bla</i><sub>CTX-M</sub> and <i>bla</i><sub>TEM</sub>) were shown in 33.8% (25/74). The <i>bla</i><sub>CTX-M</sub> gene sequence showed homology to <i>bla</i><sub>CTX-M-15</sub> from clinical isolates. The mean multiple antibiotic resistance index (MARI) was found to be higher among ESBL <i>E. coli</i> (0.25) when compared to non-ESBL <i>E. coli</i> (0.17). Both free-range husbandry management system (<i>p</i> = 0.02, OR: 30.00, 95% CI = 1.47–611.79) and high antimicrobial usage in the last 6 months (<i>p</i> = 0.01, OR: 25.17, 95% CI = 1.81–348.71) were found significantly associated with isolation of ESBL-producing <i>E. coli</i> in the tested samples using binary logistic regression analysis. This study confirmed the potential of backyard chickens as a reservoir for ESBL <i>E. coli</i> in the Jhang district, Punjab, Pakistan.
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