Fosfomycin susceptibility and molecular detection of fosA-mediated resistance among gram-negative uropathogens from a tertiary care hospital in Lucknow, India.

Urinary tract infections (UTIs) remain a major clinical challenge due to the increasing prevalence of multidrug-resistant (MDR) and extensively drug-resistant (XDR) Gram-negative uropathogens. Fosfomycin has regained importance as a therapeutic option against resistant urinary pathogens because of its unique mechanism of action and limited cross-resistance with other antimicrobial agents. However, the emergence of resistance mechanisms, particularly plasmid-mediated fosA genes, threatens its long-term clinical utility. This study employed a combined phenotypic-genotypic approach to evaluate fosfomycin resistance through minimum inhibitory concentration (MIC) determination and molecular detection of the fosA gene among Gram-negative uropathogens isolated from patients with UTIs. This prospective observational study was conducted from June 2023 to December 2024 at the Integral Institute of Medical Sciences and Research (IIMSR), Lucknow. A total of 2,550 urine samples were analyzed. Significant Gram-negative isolates were subjected to antimicrobial susceptibility testing (AST), extended-spectrum β-lactamase (ESBL) and carbapenem-resistant Enterobacterales (CRE) screening, and fosfomycin susceptibility testing using the Kirby-Bauer disc diffusion and agar dilution methods for MIC determination according to Clinical and Laboratory Standards Institute (CLSI) and European Committee on Antimicrobial Susceptibility Testing (EUCAST) guidelines. Fosfomycin-resistant isolates were further evaluated for the presence of the fosA gene using conventional polymerase chain reaction (PCR). Agreement between disc diffusion and MIC methods was assessed to determine diagnostic reliability. Of the 2,550 urine samples processed, 651 showed significant bacterial growth, yielding 371 Gram-negative isolates. Escherichia coli (79.5%) was the predominant pathogen. Fosfomycin demonstrated high overall susceptibility, with 91.6% susceptibility by disc diffusion and 94.8% by agar dilution. The high categorical agreement (96%) between both methods supports the utility of disc diffusion as a reliable screening method for fosfomycin susceptibility testing. Fosfomycin retained excellent activity against resistant phenotypes, including ESBL-producing (93%), MDR (94%), and CRE (89.6%) isolates. Fosfomycin MIC values ranged from 0.25 to 256 µg/mL, with most isolates showing MIC values within the susceptible range. The fosA gene was detected in 78.9% (15/19) of phenotypically fosfomycin-resistant isolates tested, predominantly among E. coli (84.6%) and Klebsiella pneumoniae (100%) isolates. Fosfomycin demonstrated preserved in vitro activity against Gram-negative uropathogens, including ESBL-producing, MDR, and CRE isolates. The detection of fosA among resistant isolates highlights the emergence of transferable fosfomycin resistance mechanisms and emphasizes the importance of continuous molecular surveillance combined with phenotypic susceptibility testing to support antimicrobial stewardship.