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Volume 28, Number 11—November 2022

Sequence-Based Identification of Metronidazole-Resistant Clostridioides difficile Isolates

Wiep Klaas SmitsComments to Author , Céline Harmanus, Ingrid M.J.G. Sanders, Lynn Bry, Grace A. Blackwell, Quinten R. Ducarmon, Eliane de Oliveira Ferreira, and Ed J Kuijper
Author affiliations: Centre for Microbial Cell Biology, Leiden, the Netherlands (W.K. Smits); Leiden University Medical Center, Leiden, the Netherlands (W.K. Smits, C. Harmanus, I.M.J.G. Sanders, Q.R. Ducarmon, E.J. Kuijper); Brigham & Women’s Hospital, Boston, Massachusetts, USA (L. Bry); Harvard Medical School, Boston (L. Bry); European Bioinformatics Institute (EMBL-EBI), Hinxton, UK (G.A. Blackwell); Wellcome Sanger Institute, Hinxton (G.A. Blackwell); Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil (E.O. Ferreira); National Institute for Public Health and the Environment, Bilthoven, the Netherlands (E.J. Kuijper)

Cite This Article


The plasmid pCD-METRO confers metronidazole resistance in Clostridioides difficile. We showed high sequence similarity among pCD-METRO plasmids from different isolates and identified pCD-METRO and associated metronidazole-resistant isolates in clinical and veterinary reservoirs in the Americas. We recommend using PCR or genomic assays to detect pCD-METRO in metronidazole-resistant C. difficile.


Comparison of pCD-METRO open reading frames and phylogenetic analysis in study of sequence-based identification of metronidazole-resistant Clostridioides difficile isolates. A) Linear maps compare the open reading frames (ORF)1–8 of the pCD-METRO reference sequence (identical to the RT005 plasmid) with variant pCD-METRO sequences, including the ST15 isolate from the United States (top). No ribotyping information was available for the ST15 isolate, but it should be noted that RT010 isolates belong to the same sequence type. Amino acid substitutions and nucleotide substitutions (in parentheses) are indicated above the ORFs. Colors indicate the location of putative mobilization genes (yellow), a replication gene (green), an integrase gene (blue), and genes encoding other functions (red) in the ORFs (3). The invariant regions are indicated by gray shading, and the binding location of the oBH1/2 primer set is shown in ORF6. The primer set is used for national sentinel surveillance and diagnostics of C. difficile infections in the Netherlands. Toxigenic RT/STs are indicated in red font and were all derived from symptomatic patients with C. difficile infections. Where available, the source (human/veterinary) is indicated. Isolate 1143 from Brazil was not included in this figure because no sequence information was available. B) Phylogenetic tree generated using IQ-TREE (10) and Roary (11) to show the relatedness between 2 RT005 patient isolates (LUMCMM20 0131 and LUMCMM20 0132) compared with the 2 reference strains LL-005 (RT005) and 630 (RT012). The tree is rooted on strain 630, and RT005 isolates are highlighted in yellow. Only the LUMCMM20 0132 isolate was positive for pCD-METRO. Scale bar indicates nucleotide substitutions per site. RT, ribotype; ST, sequence type.

Figure. Comparison of pCD-METRO open reading frames and phylogenetic analysis in study of sequence-based identification of metronidazole-resistant Clostridioides difficileisolates. A) Linear maps compare the open reading frames (ORF)1–8 of...

Clostridioides difficile is a major cause of antibiotic-associated 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 Study

Since 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.

Among C. difficile isolates from the Netherlands, we identified a toxigenic pCD-METRO–positive isolate (LUMCMM20 0132, National Center for Biotechnology Information [NCBI] BioSample no. SAMN26573026) from a symptomatic patient with C. difficile infection. The isolate belonged to RT005, a ribotype not reported previously to carry pCD-METRO. RT005 accounts for ≈4% of C. difficile isolates in Europe (7) and shows a similar prevalence in the Netherlands. The patient did not respond to metronidazole treatment, and a metronidazole Etest on BBA, performed at Leiden University Medical Center, confirmed the isolate was metronidazole-resistant (MIC = 8 mg/L). In contrast, a plasmid-negative RT005 isolate obtained earlier from the same patient (LUMCMM20 0131, NCBI BioSample no. SAMN26573027) was metronidazole-susceptible (MIC = 0.125 mg/L), further suggesting acquired resistance after pCD-METRO acquisition. Illumina whole-genome sequencing (NCBI BioProject accession no. PRJNA814863) and analysis of draft genomes using Kbase (8) indicated LUMCMM20 0131 and LUMCMM20 0132 were highly homologous, had an average nucleotide identity (ANI) of >99.99%, and were categorized as sequence type 6, clade 1 (9). We performed phylogenomic analysis by using IQ-TREE (10) and Roary (11) to show the 2 patient isolates were distinct from the RT005 reference strain LL005 (ANI 99.91–99.92) and RT012 reference strain 630 (ANI 99.16–99.18) (Figure, panel B). Moreover, we identified only 1 single-nucleotide polymorphism (SNP) when we aligned reads from LUMCMM20 0132 in a reference assembly against the draft LUMCMM20 0131 genome (minimum coverage 10, minimum variant frequency 0.8). We revealed that differences in the 2 patient isolates were driven by pCD-METRO carriage in LUMCMM20 0132 in a pangenome analysis using Kbase (8). We identified the pCD-METRO contig in the draft genome by using a homology search, removed terminal repeats, and circularized the sequences by using Geneious R9.1 ( The resulting plasmid sequence was 100% identical to the pCD-METRO reference sequence (GenBank accession no. OM972905) (Figure, Panel A), which likely explains the metronidazole-resistant phenotype.

Because the presence of pCD-METRO is rare, we identified pCD-METRO–positive isolates in public repositories. We queried a curated database of >661,000 assembled bacterial genomes (12) by using a compact bit-sliced signature index with a k-mer similarity threshold of 0.4. A total of 465 assemblies were returned, but only 1 C. difficile isolate had a close-hit of 0.99 k-mer similarity. The other hits had k-mer similarities of <0.49 and included different species. The C. difficile isolate containing a contig with sequence homology to pCD-METRO was V356 (NCBI BioSample no. SAMN08813897). V356 is a nontoxigenic sequence type 15 isolate cultured from an intensive care unit patient in the United States who was an asymptomatic C. difficile carrier; the isolate clustered with other nontoxigenic C. difficile genomes (13). The isolate was metronidazole-resistant (MIC = 16–24 mg/L) in an Etest on BBA medium (the test was performed at Brigham and Women’s Hospital at the time of identification). We assembled the whole-genome sequence of the isolate by using Kbase (8) and reconstructed the pCD-METRO plasmid from the draft genome sequence as described above. The plasmid had 2 SNPs compared with the pCD-METRO reference sequence: G5441C, resulting in a Q96E amino acid substitution in the ORF7 hydrolase protein, and C5904A upstream of ORF7 (Figure, panel A); other variants are described elsewhere (3). V356 extends the geographic range of pCD-METRO and associated plasmid-mediated metronidazole resistance to North America.

To facilitate homology-based identifications, we deposited a pCD-METRO sequence assembly (GenBank accession no. OM972905) for inclusion in databases of antimicrobial resistance and mobilization determinants, such as the Comprehensive Antibiotic Resistance Database (14) and PlasmidFinder (15). The deposited file also indicates the sequence variants described in this study.


SNPs in pCD-METRO have been reported in ORF1, the ORF6–ORF7 intergenic region, ORF7, and ORF8, but not in the region that contains ORF2–6; major deletions or rearrangements in this plasmid have not been found. Thus, PCR-based approaches that detect conserved plasmid regions and genomic methods that examine pCD-METRO sequences can be used to identify pCD-METRO–containing C. difficile isolates. Of note, all isolates that carried pCD-METRO were confirmed to be metronidazole-resistant (MIC >2 mg/L) in susceptibility tests. Whereas the sequences responsible for metronidazole resistance in pCD-METRO have not yet been identified, we show that the presence of pCD-METRO in C. difficile predicts metronidazole resistance. We suggest using the invariant ORF2–6 region for PCR-based detection of pCD-METRO.

We found pCD-METRO in a metronidazole-resistant toxigenic RT005 isolate from the Netherlands and also identified pCD-METRO–associated metronidazole resistance in C. difficile isolates from North and South America. We recommend using sequence-based molecular approaches to detect pCD-METRO for plasmid-mediated metronidazole-resistant C. difficile.

Dr. Smits is an associate professor in medical microbiology at Leiden University Medical Center. His research interests focus on antimicrobial drugs, antimicrobial resistance, and plasmids of C. difficile.



We thank S. Nijssen for submitting fecal samples to the National Reference Laboratory for C. difficile, M. Delaney and K. Rainha for MIC testing, and members of the Leiden University Medical Center experimental bacteriology research group for useful discussions.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Genome sequencing and susceptibility testing at Brigham & Women’s Hospital was supported by the Hatch Family Foundation and US National Institutes of Health grant nos. R01 AI153605 and P30 DK034854.

The opinions expressed by the authors do not necessarily reflect the opinions of the institutions with which the authors are affiliated.



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Cite This Article

DOI: 10.3201/eid2811.220615

Original Publication Date: October 13, 2022

Table of Contents – Volume 28, Number 11—November 2022

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Wiep Klaas Smits, Department of Medical Microbiology, Leiden University Medical Center, PO Box 9600, 2300RC, Leiden, the Netherlands

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Page created: September 16, 2022
Page updated: October 24, 2022
Page reviewed: October 24, 2022
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