Chromosomal Resistance to Metronidazole inClostridioides difficilecan be Mediated By Epistasis Between Iron Homeostasis and Oxidoreductases

Abstract

25Chromosomal resistance to metronidazole has emerged in clinical Clostridioides 26 difficile, but the genetic mechanisms remain unclear. This is further hindered by the 27 inability to generate spontaneous metronidazole-resistant mutants in the lab to aid 28 genetic studies. We therefore constructed a mismatch repair mutator, in non-toxigenic 29 ATCC 700057, to unbiasedly survey the mutational landscape for de novo resistance 30 mechanisms. In separate experimental evolutions, the mutator adopted a deterministic 31 path to resistance, with truncation of ferrous iron transporter FeoB1 as a first-step 32 mechanism of low level resistance. Allelic deletion of feoB1 in ATCC 700057 reduced 33 intracellular iron content, appearing to shift cells toward flavodoxin-mediated 34 oxidoreductase reactions, which are less favorable for metronidazole's cellular action. 35Higher level resistance evolved from sequential acquisition of mutations to catalytic 36 domains of pyruvate-ferredoxin oxidoreductase (PFOR encoded by nifJ); a synonymous 37 codon change to xdhA1 (xanthine dehydrogenase subunit A), likely affecting its 38 translation; and lastly, frameshift and point mutations that inactivated the iron-sulfur 39 cluster regulator (IscR). Gene silencing of nifJ, xdhA1 or iscR with catalytically dead 40Cas9 revealed that resistance involving these genes only occurred when feoB1 was 41 inactivated i.e. resistance was only seen in an feoB1-deletion mutant and not the 42 isogenic wild-type parent. These findings show that metronidazole resistance in C. 43 difficile is complex, involving multi-genetic mechanisms that could intersect with iron-44 dependent metabolic pathways. 45 47Clostridioides difficile infection (CDI) is a leading cause of diarrhea in hospitalized 48 patients in developed countries. Since 2003, the emergence and spread of epidemic 49 strains has significantly increased the incidence and severity of CDI. The health care 50

A superoxide reductase contributes toClostridioides difficileresistance to oxygen

“…Sensitivity to multiple antibiotics was not altered due to SOR disruption, but sensitivity to metronidazole, cefoxitin and trimethoprim was increased in both strains following oxygen exposure. Oxygenation has been shown to enhance the efficacy of the antibiotic amikacin in killing Mycobacterium abscessus (56), and in C. difficile , resistance to metronidazole has been linked to mutations in the pyruvate-ferredoxin/flavodoxin oxidoreductase gene ( nifJ ) (57), though the link between oxygen stress response and antibiotic sensitivity is not well understood.…”

Decoding a cryptic mechanism of metronidazole resistance among globally disseminated fluoroquinolone-resistant Clostridioides difficile

“…Metronidazole is a nitroimidazole prodrug that is activated by cellular reduction to anion and nitroso intermediates that reacts with DNA and proteins and depletes cellular thiols 21 .…”

“…Metronidazole is a nitroimidazole prodrug that is activated by cellular reduction to anion and nitroso intermediates that reacts with DNA and proteins and depletes cellular thiols 21 . Herein, we describe the discovery and genetic validation of the mechanism for heme-dependent resistance to metronidazole, followed by revealing its role in the global transmission of epidemic C. difficile through genome-wide association studies and population genetics.…”

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 2dehydrogenase, and glucose 6-dehydrogenase, which are related to the growth of C. difficile [50] [51], and xanthine dehydrogenase, which is related to the high-level resistance of C. difficile to antibiotics treatments [52].…”