Chromosomal Resistance to Metronidazole in Clostridioides difficile Can Be Mediated by Epistasis between Iron Homeostasis and Oxidoreductases

Chromosomal resistance to metronidazole has emerged in clinical Clostridioides difficile isolates, but the genetic mechanisms remain unclear. This is further hindered by the inability to generate spontaneous metronidazole-resistant mutants in the lab to interpret genetic variations in clinical isolates. We therefore constructed a mismatch repair mutator in nontoxigenic ATCC 700057 to survey the mutational landscape for de novo resistance mechanisms. In separate experimental evolutions, the mutator adopted a deterministic path to resistance, with truncation of the ferrous iron transporter FeoB1 as a first-step mechanism of low-level resistance. Deletion of feoB1 in ATCC 700057 reduced the intracellular iron content, appearing to shift cells toward flavodoxin-mediated oxidoreductase reactions, which are less favorable for metronidazole's cellular action. Higher-level resistance evolved from sequential acquisition of mutations to catalytic domains of pyruvate-ferredoxin/flavodoxin oxidoreductase (PFOR; encoded by nifJ), a synonymous codon change to putative xdh (xanthine dehydrogenase; encoded by CD630 31770), likely affecting mRNA stability, and last, frameshift and point mutations that inactivated the iron-sulfur cluster regulator (IscR). Gene silencing of nifJ, xdh, or iscR with catalytically dead Cas9 revealed that resistance involving these genes occurred only when feoB1 was inactivated; i.e., resistance was seen only in the feoB1 deletion mutant and not in the isogenic wild-type (WT) parent. Interestingly, metronidazole resistance in C. difficile infection (CDI)-associated strains carrying mutations in nifJ was reduced upon gene complementation. This observation supports the idea that mutation in PFOR is one mechanism of metronidazole resistance in clinical strains. Our findings indicate that metronidazole resistance in C. difficile is complex, involving multigenetic mechanisms that could intersect with iron-dependent and oxidoreductive metabolic pathways.