Sequence-Based Identification of Metronidazole-Resistant Clostridioides difficile Isolates

Abstract

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.

Keywords: Antimicrobial resistance; Brazil; Clostridioides difficile; South America; United States; bacteria; enteric infections; metronidazole; pCD-METRO; plasmid; sequence analysis; the Netherlands.

Figure

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.