Glyphosate, a herbicide, and fosfomycin, an antibiotic in clinical use- evidence of common selectable genotypes

The emergence of antimicrobial resistance (AMR) is increasingly linked to metabolic adaptation, yet the evolutionary routes underlying cross-resistance between structurally related compounds remain poorly understood. Here, whole genome sequencing (WGS) was used to analyse Klebsiella pneumoniae mutants evolved under sub-lethal glyphosate (GLP) or fosfomycin (FOS) exposure to determine how these stresses shape resistance and physiology. Sub-lethal GLP exposure increased FOS resistance, demonstrating cross-resistance between the two phosphonates. FOS-evolved mutants achieved high-level resistance through the accumulation of multiple mutations affecting the antibiotic target MurA, transport systems, and global metabolic regulation, producing a layered FOS resistance phenotype. In contrast, GLP-evolved mutants acquired similar functional classes of mutations but exhibited lower baseline FOS resistance, suggesting trade-offs between resistance and metabolic fitness. Further, analysis of FOS-evolved and GLP-evolved mutants across known bacterial GLP resistance mechanisms demonstrated a strong overlap. Comparative genomic analysis revealed a small, recurrent set of genes under selection in both evolutionary trajectories, with identical genomic loci repeatedly targeted, consistent with convergent evolution. Many of these changes were linked to central metabolism, redox balance, and cell surface regulation. For some isolates, a hypermutator phenotype was necessary to offset the potentially lethal effects of primary-target mutations through compensatory genomic adaptation. In conclusion, GLP and FOS select for shared adaptive networks that couple metabolic rewiring with AMR, revealing cross-resistance as an emergent property of global physiological reprogramming and providing mechanistic insight into ecological models of co-selection in environmental systems. Importance statementGlyphosate is a herbicide in current use worldwide. Its impact on the susceptibility of bacteria to antibiotics, remains to be described. This manuscript details a critical phenotypic and genomic analysis of shared resistance mechanisms between glyphosate and the antibiotic fosfomycin. Using Klebsiella pneumoniae, a zoonotic pathogen, this manuscript demonstrates that evolutionary adaptation to either compound results in a substantial overlap in gene mutations. Crucially, this manuscript shows that exposure to sub-lethal concentrations of glyphosate can increase resistance to fosfomycin. These findings reveal a link between agricultural chemical use and the emergence of cross-resistance to fosfomycin. By highlighting how environmental factors drive antimicrobial resistance, this study underscores an urgent need for revision of food safety and regulatory frameworks to protect One Health. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=185 SRC="FIGDIR/small/725383v1_ufig1.gif" ALT="Figure 1"> View larger version (46K): org.highwire.dtl.DTLVardef@d44420org.highwire.dtl.DTLVardef@1c4a5d1org.highwire.dtl.DTLVardef@1624052org.highwire.dtl.DTLVardef@16d08d4_HPS_FORMAT_FIGEXP M_FIG C_FIG