The Metabolic State of E. coli Influences Fosfomycin Efficacy and Promotes Resistance Evolution

The phosphonic antibiotic fosfomycin is a bacterial cell wall synthesis inhibitor that targets MurA, the first enzyme in the peptidoglycan pathway. Transporter loss or enzymatic inactivation confers resistance to fosfomycin, but whether the metabolic state of a bacterium influences the efficacy of this antibiotic has not been characterized. Here, we used an Escherichia coli CRISPR interference library targeting 1,515 metabolic genes to identify metabolic activities that influence fosfomycin efficacy. We discovered that knockdowns of ATP synthase and pyruvate kinase genes lead to a regrowth phenotype, whereby cells resume growth after an initial phase of killing. By following up on this phenotype with population analysis profile tests and repeated treatment cycles, we found evidence that a heteroresistant population may promote the evolution of fosfomycin resistance. Whole-genome sequencing of the pykF CRISPRi strain after 24 h of fosfomycin exposure revealed that the acid stress protein-encoding gene ibaG, which is upstream of murA, carries a mutation that confers fosfomycin resistance. Metabolome analysis showed accumulation of the MurA substrate phosphoenolpyruvate in regrowing cells, which may compete with fosfomycin for binding to MurA. Transcriptome analysis provided further insight into the mechanism of cell regrowth, including upregulation of genes encoding cell envelope stress response regulators such as cpxP. These results suggest that the metabolic state can modulate the efficacy of fosfomycin and contribute to resistance evolution.