Simple SummaryEndometritis, one of the leading causes of mares’ infertility and likely originates from bacteria inhabiting the vaginal microflora which ascend the genitourinary tract. Urogenital infections are caused by opportunistic or commensal microorganisms, and among themEscherichia colistrains have frequently been isolated and associated with mare infertility. Herein, we report the phenotypic and genotypic characterization of 24E. colistrains isolated from the uterus of 24 independent mares which had been examined by an equine veterinarian because of fertility problems. Antimicrobial-resistance, biofilm production, the ability to adhere and invade HeLa cells as well as the carriage of specificE. coliassociated virulence genes were investigated. Particular attention was given to the profiles ofE. colistrains isolated by direct-plating and those that required enrichment in broth before seeding on the plate.AbstractEscherichia coliis the bacterial pathogen most frequently associated with mare infertility. Here, we characterized 24E. colistrains isolated from mares which presented signs of endometritis and infertility from a genotypic and phenotypic point of view. The majority of the isolates belonged to phylogenetic group B1 (9/24, 37.5%). Regarding antibiotic resistance profiles, 10 out of 24 (41.7%) were multidrug-resistant (MDR). Moreover, 17 out of 24 (70.8%) were strong or moderate biofilm producers, and of these eight were MDR strains. Interestingly, 21 out of 24 (87.5%)E. colistrains were phenotypically resistant to ampicillin and 10 of them were also resistant to amoxicillin with clavulanic acid. Regarding the presence of selected virulence factors, 50% of the examined strains carried at least three of them, withfimHdetected in all strains, and followed bykpsMTII(11/24, 45.9%). No strain was able to invade HeLa cell monolayers. No relevant differences for all the investigated characteristics were shown by strains that grew directly on platesversusstrains requiring the broth-enrichment step before growing on solid media. In conclusion, this work provides new insight intoE. colistrains associated with mares’ infertility. These results broaden the knowledge ofE. coliand, consequently, add useful information to improve prevention strategies and therapeutic treatments contributing to a significant increase in the pregnancy rate in mares.