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 using molecularly intact heme. Here, we describe the mechanism underlying this trait. We find that most metronidazole-resistant C. difficile strains carry a T-to-G mutation (which we term PnimBG) in the promoter of gene nimB, resulting in constitutive transcription. Silencing or deleting nimB eliminates metronidazole resistance. NimB is related to Nim proteins that are known to confer resistance to nitroimidazoles. We show that NimB is a heme-dependent flavin enzyme that degrades nitroimidazoles to amines lacking antimicrobial activity. Furthermore, occurrence of the PnimBG mutation is associated with a Thr82Ile substitution in DNA gyrase that confers fluoroquinolone resistance in epidemic strains. Our findings suggest that the pandemic of fluoroquinolone-resistant C. difficile occurring over the past few decades has also been characterized by widespread resistance to metronidazole.

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

“…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].…”

“…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].…”

Proteomics-Based RT-qPCR and Functional Analysis of 18 Genes in Metronidazole Resistance of Bacteroides fragilis

“…The Nim enzymes are nitro-reductases that can transfer either six [ 6 ] or two electrons to the nitro group of metronidazole, yielding either an amino or a nitroso imidazole, respectively [ 20 ]. Recently, in vivo and in vitro experiments demonstrated that a nim group enzyme encoded by Clostridioides difficile strains is a nitro-reductase [ 7 ]. In this latter study, it was also confirmed that metronidazole resistance in C. difficile is dependent on hemin [ 32 ] through experiments involving the direct addition of metronidazole to assay its modification by recombinant NimB and by transcriptomic analysis.…”

“…The best known and most investigated metronidazole resistance mechanism of BFG strains is mediated by the nim genes, a few of which were originally discovered in the 1980s and 1990s [ 5 ]. There are now 12 known homologs of nim (i.e., nimA-L ) that share 50–80% amino acid homologies, and they are all proposed to act as nitro-reductases [ 6 , 7 ]. They have been localized to both the plasmid and chromosome, and they all require an upstream copy of a Bacteroides -specific insertion sequence (IS) element with promoter sequences to function in metronidazole resistance [ 5 ].…”

Antibacterial activity of ibezapolstat against antimicrobial-resistant clinical strains of Clostridioides difficile

“…Only two antibiotics, vancomycin (VAN) and fidaxomicin (FDX), are guidelines recommended for C. difficile infection (CDI) ( 2 ), and antimicrobial resistance has been reported to both ( 3 , 4 ). A third antibiotic, metronidazole (MTZ), is no longer a guideline recommended due to decreasing clinical response rates most likely due to antimicrobial resistance ( 5 , 6 ). Although novel therapeutics are in development for CDI, including the recent approval of live biotherapeutic products, only three antibiotics have made it to phase III trials since the approval of FDX in 2011, and none have filed for Food and Drug Administration approval ( 7 – 12 ).…”