Chromosomal Resistance to Metronidazole in Clostridioides difficile can be Mediated By Epistasis Between Iron Homeostasis and Oxidoreductases

Abstract

Chromosomal resistance to metronidazole has emerged in clinical Clostridioides difficile, but the genetic mechanisms remain unclear. This is further hindered by the inability to generate spontaneous metronidazole-resistant mutants in the lab to aid genetic studies. We therefore constructed a mismatch repair mutator, in non-toxigenic ATCC 700057, to unbiasedly survey the mutational landscape for de novo resistance mechanisms. In separate experimental evolutions, the mutator adopted a deterministic path to resistance, with truncation of ferrous iron transporter FeoB1 as a first-step mechanism of low level resistance. Allelic deletion of feoB1 in ATCC 700057 reduced intracellular iron content, appearing to shift cells toward flavodoxin-mediated oxidoreductase reactions, which are less favorable for metronidazoles cellular action. Higher level resistance evolved from sequential acquisition of mutations to catalytic domains of pyruvate-ferredoxin oxidoreductase (PFOR encoded by nifJ); a synonymous codon change to xdhA1 (xanthine dehydrogenase subunit A), likely affecting its translation; and lastly, frameshift and point mutations that inactivated the iron-sulfur cluster regulator (IscR). Gene silencing of nifJ, xdhA1 or iscR with catalytically dead Cas9 revealed that resistance involving these genes only occurred when feoB1 was inactivated i.e. resistance was only seen in an feoB1-deletion mutant and not the isogenic wild-type parent. These findings show that metronidazole resistance in C. difficile is complex, involving multi-genetic mechanisms that could intersect with iron-dependent metabolic pathways.