Genomic analysis of Fosfomycin resistance in multi-drug resistant uropathogens and comparison of in-vitro susceptibility methods uropathogens

Background and Objectives: Urinary tract infection is one of the most common bacterial infections causing high morbidity and mortality. Thealarming rise of multidrug-resistant uropathogens worldwide forced the clinician to re- think 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 againstdifferent drug-resistant uropathogens. Materials and Methods: Consecutive 181 uropathogens were tested for Fosfomycin susceptibility using agar dilution, disc diffusion and E-test. Resultswere 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 thegold 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 diffu- sion for all the organisms were91.2%/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 Fos- fomycin 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 Fosfo- mycin susceptibility testing compared to agar dilution.