| Summary: | Background: Pyometra is a diestrual chronic disease frequently associated with <i>Escherichia coli</i>. Initial pyometra treatment involves empiric antimicrobial therapy whose suitability should be confirmed by antimicrobial susceptibility testing. Antimicrobial resistance is a major health issue for veterinary medicine, rendering surveillance studies essential. Our goal was to determine the susceptibility profile of <i>E. coli</i> isolates obtained from healthy and pyometra-presenting dogs and to compare the application of different antimicrobial susceptibility guidelines. Methods: The antimicrobial susceptibility profile (ASP) of 74 <i>E. coli</i> isolates was determined by disk diffusion, using six antimicrobials commonly used in veterinary medicine. Profiles were assessed by CLSI VET01S, CLSI M100 and EUCAST guidelines. β-lactamases-encoding genes <i>bla</i><sub>TEM</sub>, <i>bla</i><sub>SHV</sub> and <i>bla</i><sub>OXA</sub> were detected by multiplex PCR. Biofilm production ability was evaluated by pellicle formation assays in Luria–Bertani medium. Results: Variations in the resistance frequency were observed for amoxicillin/clavulanic acid, cephalexin and cefotaxime (29.7–54.1%, 10.8–16.2% and 1.4–4.1%, respectively). Results varied slightly between clinical and commensal isolates, as well as their biofilm-forming ability. Genes <i>bla</i><sub>TEM</sub>, <i>bla</i><sub>SHV</sub> and <i>bla</i><sub>OXA</sub> were detected in 25.5%, 11.8% and 9.8% of isolates, respectively. Conclusions: Results show the importance of ASP determination in veterinary isolates and the need for using standardized and validated testing methods and harmonized interpretive criteria.
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