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 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.

Haem is crucial for medium-dependent metronidazole resistance in clinical isolates of Clostridioides difficile

“… 14 More recently, a mutator strain defective in DNA mismatch repair was evolved in the presence of metronidazole. 15 The study discovered mutations in a gene encoding an iron transporter ( feoB1 ) in metronidazole-resistant strains and showed that sequential mutations in nifJ [encoding the pyruvate-ferredoxin oxidoreductase (‘PFOR’)], xdh (encoding xanthine dehydrogenase) or iscR (encoding an iron–sulphur cluster regulator) could further increase metronidazole resistance. 15 Though studies with laboratory-evolved strains are informative, it is unclear how these findings translate to metronidazole-resistant strains isolated from subjects outside the laboratory.…”

Impact of subinhibitory concentrations of metronidazole on proteome of Clostridioides difficile strains with different levels of susceptibility

“…Peak lists were generated as mzXML files using MSConvert software (ProteoWizard 3.0). Mass spectrometric data was searched using X!TandemPipeline software version 0.2.38 developed by PAPPSO (Plateforme d’Analyse Protéomique de Paris Sud Ouest) facility [ 31 ]. Peptide searches were performed with the following parameters: enzymatic cleavage by trypsin digestion with one possible mis-cleavage, precursor mass tolerance of ±10 ppm and fragment mass tolerance of 0.5 Da, static modifications of carbamidomethylated cysteine and potential modification of oxidized Methionine.…”

“…Higher-level resistance evolved from sequential acquisition of mutations to catalytic domains of pyruvate-ferredoxin/flavodoxin oxidoreductase (PFOR), a synonymous codon change to putative xdh (xanthine dehydrogenase; encoded by CD630_31770 ), and frameshift and point mutations that inactivated the iron-sulfur cluster regulator ( iscR ). However, resistance involving these genes was seen only in the feoB1 deletion mutant and not in the isogenic parental strain [ 31 ].…”

Forward variable selection improves the power of random forest for high-dimensional microbiome data

“…Conversely, some of the functional profiles were strongly enriched in the CDI cases. They included alcohol dehydrogenase, L-iditol 2-dehydrogenase, and glucose 6-dehydrogenase, which are related to the growth of C. difficile [48,49], and xanthine dehydrogenase, which is related to the high-level resistance of C. difficile to antibiotics treatments [50].…”