Characterization of fosS, a novel fosfomycin-modifying enzyme gene identified in Staphylococcus haemolyticus

Abstract The efficacy of fosfomycin is increasingly challenged by emerging resistance mechanisms, primarily involving fosfomycin-modifying enzymes and target (MurA) alterations. This study aimed to elucidate the novel mechanism underlying fosfomycin resistance in fosfomycin-resistant Staphylococcus haemolyticus (FRSH) strains. Whole-genome sequencing and bioinformatic analysis identified a novel fosfomycin-modifying enzyme, designated FosS , and new mutations (D290H/G359S) on MurA. Cloning experiments further demonstrated that the expression of fosS increased the minimum inhibitory concentration of fosfomycin 32-fold in S. aureus RN4220, whereas MurA mutations induced a 4-fold increase in minimum inhibitory concentration. Phylogenomic analysis revealed that fosS -positive S. haemolyticus (FPSH) is globally distributed, forming a distinct lineage across swine-derived samples from South Korea, South Africa, and China. Moreover, mobile genetic element IS Enfa4 , which facilitated horizontal transfer of cfr, was detected downstream of fosS . Notably, various antimicrobial resistance genes (ARGs) were also detected in FPSH. Although fosfomycin was forbidden in food animals in China, the fitness advantages can be conferred by the other ARGs under antimicrobial selective pressure and may facilitate the transmission of FPSH. Overall, this study is the first to report a novel fosfomycin resistance gene and new MurA variants in FRSH. The IS Enfa4 was downstream of fosS and underscored potential mobilization risks. These findings provided a new perspective on One Health to control fosfomycin resistance transmission.