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Objectives: To evaluate the occurrence of plasmid-mediated fos genes among fosfomycin-resistant Escherichia coli isolates collected from patients in Lisbon, Portugal, and characterize the fos-positive strains., Methods: A total of 19 186 E. coli isolates were prospectively collected between April 2022 and January 2023 from inpatients and outpatients at a private laboratory in Lisbon. Fosfomycin resistance was initially assessed by semi-automated systems and further confirmed by the disc diffusion method. Resistant isolates were investigated for plasmid-mediated fos genes (fosA1-fosA10, fosC and fosL1-fosL2) and extended-spectrum beta-lactamases (ESBLs) by PCR and sequencing. Multilocus sequence typing was performed to evaluate the clonal relationship among fos-carrying isolates., Results: Out of the 19 186 E. coli isolates, 100 were fosfomycin-resistant (0.5%), out of which 15 carried a fosA-like gene (15%). The most prevalent fosfomycin-resistant determinant was fosA3 (n = 11), followed by fosA4 (n = 4). Among the 15 FosA-producing isolates, 10 co-produced an ESBL (67%), being either of CTX-M-15 (n = 8) or CTX-M-14 (n = 2) types. The fosA3 gene was carried on IncFIIA-, IncFIB-, and IncY-type plasmids, whereas fosA4 was always located on IncFIB-type plasmids. Most FosA4-producing isolates belonged to a single sequence type ST2161, whereas isolates carrying the fosA3 gene were distributed into nine distinct genetic backgrounds., Conclusion: The prevalence of fosfomycin-resistant E. coli isolates is still low in Portugal. Notably, 15% of fosfomycin-resistant isolates harbour a transferable fosA gene, among which there is a high rate of ESBL producers, turning traditional empirical therapeutical options used in Portugal (fosfomycin and amoxicillin-clavulanic acid) ineffective., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)