Background: Due to the increase of multidrug resistance (MDR) in organisms that cause urinary tract infections (UTIs), we need to investigate anti-microbial resistance (AMR) in these pathogens on a regular basis. Objectives: The main purpose of this cross-sectional study was the use of updated phylotyping method to evaluate the possible correlations between biofilm production, hemolysin production, and antibiotic resistance among uropathogenic Escherichia coli (UPEC). Materials and Methods: A total of 138 UPEC isolates were evaluated for biofilm formation, hemolysin production, and antimicrobial susceptibility to five classes of antibiotics, including quinolones, β-lactams, tetracyclines, and sulfonamides. The phylogenetic structure was determined using the original and recently updated protocols. Results: Our results demonstrated that of 138 UPEC isolates, the majority belonged to phylogenetic group B2 (34.7%), followed by F (13.7%). Ninety-four (68%) isolates showed hemolytic activity but hemolysis had no correlation with antibiotic resistance while a correlation was observed between the hemolytic activity and biofilm formation. Moderate to strong biofilm production was observed in 34.7% of the isolates. Additionally, 73% of them showed hemolytic activity and most of them belonged to B2 phylogroup (37.5%). In this study, increasing rates of phylogroup F were detected compared to the old method that indicates the possible importance of this phylotype in UTI. Additionally, detecting the novel phylogroup G provides more precise data which can only be obtained by the new method. Conclusion: The findings of the present study showed that more precise phylotyping results can be obtained when evaluating different aspects of UPEC in epidemiological studies using the new complementary method.

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