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

## TL;DR

This study shows that the metabolic state of E. coli affects how well fosfomycin works and can lead to antibiotic resistance.

## Contribution

The study reveals how specific metabolic gene knockdowns influence fosfomycin efficacy and resistance evolution in E. coli.

## Key findings

- Knockdowns of ATP synthase and pyruvate kinase genes lead to regrowth after initial killing by fosfomycin.
- The gene ibaG, upstream of murA, carries a mutation conferring fosfomycin resistance.
- Regrowing cells accumulate phosphoenolpyruvate, which may compete with fosfomycin for MurA binding.

## Abstract

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.