Tehran University of Medical Sciences Iranian Journal of Microbiology 2008-3289 14 5 2022 10 22 636 644 Raunak Bir Department of Microbiology, All India Institute of Medical Sciences, New Delhi, India Sarita Mohapatra Department of Microbiology, All India Institute of Medical Sciences, New Delhi, India Arvind Kumar Department of Medicine, All India Institute of Medical Sciences, New Delhi, India Nazneen Arif Department of Microbiology, All India Institute of Medical Sciences, New Delhi, India Sonu Tyagi Department of Microbiology, All India Institute of Medical Sciences, New Delhi, India Aswin AK Department of Medicine, All India Institute of Medical Sciences, New Delhi, India Hitender Gautam Department of Microbiology, All India Institute of Medical Sciences, New Delhi, India Seema Sood Department of Microbiology, All India Institute of Medical Sciences, New Delhi, India Bimal Das Department of Microbiology, All India Institute of Medical Sciences, New Delhi, India Arti Kapil Department of Microbiology, All India Institute of Medical Sciences, New Delhi, India 2022 01 02 2022 08 08 Background and Objectives: Urinary tract infection is one of the most common bacterial infections causing high morbidity and mortality. The alarming rise of multidrug-resistant uropathogens worldwide forced the clinician to rethink the old drugs like Fosfomycin for its therapeutic management. Our objective was to compare agar dilution, disc diffusion and E-test method for antimicrobial susceptibility testing of Fosfomycin against different drug-resistant uropathogens. Materials and Methods: Consecutive 181 uropathogens were tested for Fosfomycin susceptibility using agar dilution, disc diffusion and E-test. Results were interpreted using Clinical and Laboratory Standards Institute (CLSI) and European Committee on Antimicrobial Susceptibility Testing (EUCAST) breakpoints. Whole genome sequencing analysis was done on the 4 XDR/PDR Fosfomycin resistant Klebsiella pneumoniae isolates. Results: Escherichia coli was found as the most common (62.4%) uropathogen followed by Klebsiella pneumoniae (21%). Considering agar dilution as the gold standard, 6.1% of isolates were resistant to Fosfomycin. Following CLSI breakpoints, the susceptibility of Escherichia coli, Klebsiella pneumoniae, other Enterobacterales and Pseudomonas aeruginosa were 92.9%, 92.1%, 100%, 100%; whereas using EUCAST breakpoints the susceptibility rates were 85.7%, 86.9%, 92.9%, and 100%, respectively. The essential agreement, categorical agreement, major error, and very major error for E-test/ disc diffusion for all the organisms were 91.2%/Not Applicable, 95%/93.9%, 1.8%/4.7%, 9.1%/9.1%, respectively. Whole-genome sequencing showed mutation UhpT gene as well as the presence of plasmid-mediated fosA5 or fosA6 genes conferring Fosfomycin resistance. Conclusion: This result supports very low resistance of Enterobacterales against Fosfomycin; hence should be considered a valuable option to treat multidrug-resistant uropathogens. Disc diffusion was observed to be a convenient method for Fosfomycin susceptibility testing compared to agar dilution. https://ijm.tums.ac.ir/index.php/ijm/article/view/3500 https://ijm.tums.ac.ir/index.php/ijm/article/download/3500/1500