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

Abstract

Severe outbreaks and deaths have been linked to the emergence and global spread of fluoroquinolone-resistant Clostridioides difficile over the past two decades. At the same time, metronidazole, a nitro-containing antibiotic, has shown decreasing clinical efficacy in treating C. difficile infection (CDI). Most metronidazole-resistant C. difficile exhibit an unusual resistance phenotype that can only be detected in susceptibility tests utilizing molecularly intact heme. Here we describe the mechanism underlying this trait, which we discovered using molecular genetics, phylogenetics, and population analyses. Most metronidazole-resistant strains evolved a T to G mutation, we term PnimBG, in the -10 regulatory promoter of the 5-nitroimidazole reductase nimB, resulting in the gene being constitutively transcribed. Silencing or deleting nimB eliminated metronidazole resistance. We identified the protein as a heme-dependent nitroreductase that degraded nitro-drugs to an amine lacking antimicrobial activity. We further discovered that the metronidazole-resistant PnimBG mutation was strongly associated with the Thr82Ile substitution conferring fluoroquinolone resistance in epidemic strains. Re-analysis of published genomes from global isolates confirmed that all but one encoding PnimBG also carried the Thr82Ile mutation. Our findings suggest that fluoroquinolone and metronidazole resistance co-mediated the pandemic of healthcare-associated C. difficile that are associated with poorer treatment outcomes in CDI patients receiving metronidazole.

Clostridioides difficile spores tolerate disinfection with sodium hypochlorite disinfectant and remain viable within surgical scrubs and gown fabrics

“…Patients with CDI are usually treated with antibiotics such as metronidazole, vancomycin and fidaxomicin [7]. Recent studies, however, have established antibiotic resistance within C. difficile; concerningly, plasmid-mediated metronidazole resistance (pMETRO plasmid) has been found and fluoroquinolone-resistant C. difficile has been found to be directly associated with metronidazole resistance [8,9]. Currently research into vancomycin resistance is being explored, and recently in vivo resistance to fidaxomicin has been described [10,11].…”

Characterizing metabolic drivers of Clostridioides difficile infection with activity-based hydrazine probes

“…As these Stickland reductases are cofactor-dependent, species-specific, and essential metabolic enzymes during disease, they have potential to be better pharmacological interventions in the age of antibiotic resistance. While vancomycin, fidaxomicin and metronidazole are standard antibiotic treatments for C. difficile , there is a high rate of infection reoccurrences and a high number of emerging hypervirulent strains resistant to standard care ( He et al, 2013 ; Eubank et al, 2022 ; Olaitan et al, 2022 ). In addition, these antibiotics don’t address the cause of infection—the imbalance of the microbiome—but rather combat the effects.…”

Non-Toxigenic Clostridioides difficile Strain E4 (NTCD-E4) Prevents Establishment of Primary C. difficile Infection by Epidemic PCR Ribotype 027 in an In Vitro Human Gut Model

“…Metronidazole resistance of NTCD-E4 is likely the result of SNPs in hsmA and/or a gene homologous to CD196_1331 (see above). Of note, a recent preprint provides further support for the causative involvement of, in particular, the latter mutation [ 70 ]. NTCD-E4 has other features that make it of interest as a strain for potential interventions.…”