Sequence-Based Identification of Metronidazole-Resistant Clostridioides difficile Isolates

Abstract

C lostridioides difficile is a major cause of antibioticassociated colitis (1). Antimicrobial drug-resistant infections are a global economic and healthcare burden (2). Resistance is generally low to commonly prescribed antimicrobial drugs used for primary C. difficile infections. However, high rates of metronidazole resistance have been observed for C. difficile isolates carrying the 7-kb plasmid pCD-METRO, in particular for isolates belonging to PCR ribotype (RT) 010 and RT020 (clade 1) and the epidemic strain RT027 (clade 2) (3) (Figure , panel A). This plasmid has been reported in C. difficile isolates from countries in Europe. The StudySince the discovery of pCD-METRO, we have implemented PCR that uses primers oBH-1 (5′-CCTCGTAGAATCCGGTGCAA-3′) and oBH-2 (5′-TATTTCCTTGCCGCTGAGGT-3′) for national sentinel surveillance and diagnostics of C. difficile infections in the Netherlands. The primers are specific for open reading frame (ORF) 6 of pCD-METRO (Figure , panel A). Since 2019, we have tested 3,257 isolates and identified 8 (0.25%) additional pCD-METRO-positive isolates; this percentage is consistent with previous findings (3). We have a total of 27 human and animal C. difficile isolates in our collection that are pCD-METRO-positive. Most of the isolates (22/27, 81%) belong to nontoxigenic PCR RT010, including isolate 1143 from Brazil. Isolate 1143 is one of 8 canine isolates that showed phenotypic resistance to metronidazole (MIC = 32 mg/L) by Etest on Brucella blood agar (BBA); the Etest was performed at the Universidade Federal do Rio de Janeiro in Brazil. The isolate from Brazil confirmed that pCD-METRO is present in C. difficile not only in Europe but also in South America. The 1143 isolate was not characterized further because it belonged to PCR RT010, in which pCD-METRO is most frequently observed. The high number of C. difficile RT010 isolates carrying pCD-METRO might be related to genomic background of the isolates (4) or sampling bias; a higher prevalence of metronidazole resistance has been observed among RT010 strains (3,5). Low-frequency horizontal gene transfer is more likely to occur after prolonged co-colonization of nontoxigenic C. difficile and pCD-METRO donor bacteria, and acquisition of the plasmid might occur from a source after metronidazole exposure. For example, dogs carry nontoxigenic C. difficile frequently and are often treated with metronidazole (6). The presence of pCD-METRO in toxigenic isolates might also be underestimated; antimicrobial susceptibility testing is not routinely performed, and plasmid carriage is not assessed, even when metronidazole treatment fails.

Genetic determinants of resistance to antimicrobial therapeutics are rare in publicly available Clostridioides difficile genome sequences

“…The associated resistance mechanism is not yet understood. 22 Boekhoud et al 21 confirmed the presence of pCD-METRO in several metronidazole-resistant C. difficile strains that had been reported previously. 23–25 To date, plasmid-mediated metronidazole resistance has been found in C. difficile PCR ribotypes (RTs) RT010, RT020, RT005 and in one isolate of RT027.…”

“… 23–25 To date, plasmid-mediated metronidazole resistance has been found in C. difficile PCR ribotypes (RTs) RT010, RT020, RT005 and in one isolate of RT027. 22 , 26 , 27 The majority of those isolates belonged to RT010 and were non-toxigenic. 22 Recently, a point mutation in the promoter of the nimB gene (dubbed ‘P nimB G ’) of C. difficile was reported to enhance the gene’s expression level and thereby elevate the metronidazole MIC by 4- to 8-fold.…”

“… 22 , 26 , 27 The majority of those isolates belonged to RT010 and were non-toxigenic. 22 Recently, a point mutation in the promoter of the nimB gene (dubbed ‘P nimB G ’) of C. difficile was reported to enhance the gene’s expression level and thereby elevate the metronidazole MIC by 4- to 8-fold. 28 Furthermore, truncations of chromosomal genes hsmA (exhibiting homology to haem-containing cytochromes) or feoB1 (encoding a ferrous iron transporter) were reported to cause metronidazole resistance in C. difficile.…”

“…However, these authors found no indication of fidaxomicin resistance or vancomycin resistance in their dataset. 38 Smits et al 22 screened bacterial genome sequences publicly available up to November 2018 (as provided by Blackwell et al 39 in 2021) for the pCD-METRO plasmid. Since then, however, the number of C. difficile genome sequences in the public domain has more than doubled (as of September 2023; see https://enterobase.warwick.ac.uk/species/index/clostridium ), and additional genetic determinants of antimicrobial resistance have been identified.…”

Approaching pathogenicClostridiafrom a One Health perspective

“…Groups investigating drivers of antibiotic resistance should note that the combination of genomic and phenotypic resistance data provide the most robust information to evaluate functions of genomic loci in promoting resistance, and to exercise caution in interpreting resistance gene calls in the absence of phenotypic confirmation. For example, in C. difficile AMRFinderPlus has identified plasmid-borne genes that increase strain phenotypic resistance to metronidazole 68 . In contrast, many van gene homologs that are involved in C. difficile's cell wall synthesis, promote phenotypic resistance to vancomycin in Enterococci and other species, but do not do so in C. difficile 47 .…”