One-year multicenter surveillance of Fosfomycin resistance Enterobacterales: the rise of FosA3-producing P. mirabilis

• One-year multicenter survey of fosfomycin-resistant Enterobacterales in Czech Republic hospitals. • FosA3-producing Proteus mirabilis predominates. • FosA3-positive P. mirabilis ST185 forms a local clonal cluster while E. coli clones are heterogeneous. • Transporter defects (GlpT/UhpT) are widespread; most fosA strains carry ESBL genes. • Molecular surveillance is crucial to monitor and contain emerging fosfomycin resistance. The global rise of antimicrobial resistance has renewed interest in fosfomycin (FOS), an old antibiotic with activity against multidrug-resistant Enterobacterales . However, resistance to FOS is increasing, driven by impaired drug uptake, target modification, and by fosA -encoded enzymatic inactivation. This study assessed the prevalence and molecular basis of FOS resistance among Enterobacterales collected in Czech tertiary care hospitals in 2024. A total of 211 preliminary FOS-resistant isolates were obtained from nine hospitals across the Czech Republic, predominantly Proteus mirabilis (n=149) and Escherichia coli (n=57). All isolates showed elevated FOS MICs, and the PPF test identified FosA activity in 34/211 isolates. Carbon-source growth testing demonstrated widespread impairment of GlpT and UhpT transporters (93.8% affecting both). PCR confirmed fosA genes in 14 isolates (9 P. mirabilis , 5 E. coli ). WGS revealed fosA3 as the dominant variant (85.7%), followed by fosA4 (14.3%). P. mirabilis isolates primarily belonged to ST185 and ST135, forming two Czech-specific fosA3 clusters with limited relatedness to international genomes. FosA-producing E. coli displayed broader diversity (ST69, ST58, ST550, ST1308). FosA4 was detected exclusively in E. coli . Most fosA -positive strains co-harbored ESBL genes, predominantly bla CTX-M-65 . SNP-based phylogenies indicated local clonal circulation of fosA3 -positive P. mirabilis ST185, whereas E. coli isolates showed heterogeneous international linkages. Analysis of GlpT/UhpT/MurA identified numerous amino acid substitutions, though only a minority were predicted to affect protein function. This study documents the first broader emergence of plasmid-mediated FOS resistance in Czech Enterobacterales and underscores the importance of continuous genomic surveillance of fosA -mediated resistance.