The overuse of β-lactam drugs as the primary approach to antibiotic treatment for Enterobacteria-related infections has led to the selection of β-lactam-resistant isolates, so resistance to β-lactams has become a serious concern worldwide today. This study aimed to examine the patterns of antibacterial resistance and the prevalence of extended-spectrum β-lactamases (ESBLs) genes. A total of 100 Escherichia coli isolates were collected from stool samples of children with diarrhea between December 2020 and August 2021 from three different hospitals in Mashhad, Iran. Antibacterial resistance patterns and frequency of ESBL genes including blaTEM, blaSHV, blaCTX-M and blaOXA were investigated using the Polymerase chain reaction (PCR) technique and phenotypic method. Overall, the highest resistance was towards erythromycin (100%), followed by amoxicillin-clavulanic acid (69%), trimethoprim-sulfamethoxazole (63%), and cefixime (63%). On the other hand, the highest susceptibility was detected to amikacin (98%), gentamicin (98%), nitrofurantoin (97%), and ciprofloxacin (74%), respectively. Moreover, 75% of all isolates were multi-drug resistant (MDR), and the majority of ESBL-positive iso- lates showed considerably higher resistance rates, especially to cephalosporins, compared to ESBL-negative isolates. PCR screening revealed that the most prevalent ESBL gene was blaTEM at 93%, followed by blaCTX-M at 55%, while blaOXA and blaSHV were the least common, at 10% and 6%, respectively. The results emphasized the increased number of ESBL resistance gene carriers and their global dissemination. In addition, the high rate of MDR bacteria highlights the importance of continuously assessing antimicrobials to achieve effective antibiotic treatments.

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