Genomic islands and molecular mechanisms relating to drug-resistance in Clostridioides (Clostridium) difficile PCR ribotype 176

Abstract

Objectives: To analyse characteristics of Clostridioides difficile PCR ribotype 176 clinical isolates from Poland, the Czech Republic and Slovakia with regard to the differences in its epidemiology.

Methods: Antimicrobial susceptibility testing and whole genome sequencing were performed on a selected group of 22 clonally related isolates as determined by multilocus variable-number tandem repeat analysis (n = 509). Heterologous expression and functional analysis of the newly identified methyltransferase were performed.

Results: Core genome multilocus sequence typing found 10-37 allele differences. All isolates were resistant to fluoroquinolones (gyrA_p. T82I), aminoglycosides with aac(6')-Ie-aph(2'')-Ia in six isolates. Erythromycin resistance was detected in 21/22 isolates and 15 were also resistant to clindamycin with ermB gene. Fourteen isolates were resistant to rifampicin with rpoB_p. R505K or p. R505K/H502N, and five to imipenem with pbp1_p. P491L and pbp3_p. N537K. PnimB^G together with nimB_p. L155I were detected in all isolates but only five were resistant to metronidazole on chocolate agar. The cfrE, vanZ1 and cat-like genes were not associated with linezolid, teicoplanin and chloramphenicol resistance, respectively. The genome comparison identified six transposons carrying antimicrobial resistance genes. The ermB gene was carried by new Tn7808, Tn6189 and Tn6218-like. The aac(6')-Ie-aph(2'')-Ia were carried by Tn6218-like and new Tn7806 together with cfrE gene. New Tn7807 carried a cat-like gene. Tn6110 and new Tn7806 contained an RlmN-type 23S rRNA methyltransferase, designated MrmA, associated with high-level macrolide resistance in isolates without ermB gene.

Conclusions: Multidrug-resistant C. difficile PCR ribotype 176 isolates carry already described and unique transposons. A novel mechanism for erythromycin resistance in C. difficile was identified.

Keywords: Clostridioides difficile infection; epidemiology; macrolide resistance methyltransferase; whole genome sequencing.

Figure 1.

Whole genome alignment of complete genomes of RT176 C. difficile. Nucleotide blast hits shorter than 3000 bp were omitted. Mobile genetic elements are shown and colour-coded as shown in legend.

Figure 2.

Schematic representation of the identified Tn7806 in C. difficile isolates and comparison with previously identified conjugative C. difficile transposons. a) Tn6110, which belongs to the CTn5-like elements, but also shows similarities with CTn2, both of which have been described in C. difficile strain 630. Tn7806 is identical to Tn6110 but contains two additional insertions carrying lsa-like, cfrE and aac(6´)-Ib resistance genes, respectively. The dashed line connects different parts of the same gene. b) Detail showing that the gene encoding the T4SS-ATPase VirD4 is disrupted by the insertion of Tn6105 (in Tn6110) and by the additional insertion of the cfrE-carrying insert. The insertion of the cfrE fragment leads to an in-frame fusion of virD4 with another virD4 gene (for details see Figure S4). c) Phylogenetic tree showing the relatedness of the macrolide resistance methyltransferase A (MrmA) identified in Tn6110 and Tn7806 to genomic RlmN and antimicrobial resistance Cfr 23S rRNA SAM radical methyltransferases.

Figure 3.

Schematic representation of the ermB-carrying Tn6189-derived elements. Tn6189-like and Tn7808 kb identified in this study are compared with previously identified conjugative C. difficile transposons. Tn6189 shows similarities to CTn7 and CTn1 from C. difficile strain 630. Nucleotide changes leading to premature stop codons in genes of Tn6189 are labelled.

Figure 4.

Unrooted Approximate-Maximum-Likelihood phylogenetic tree inferred with GTR model from core genome alignment (3149673 sites). C. difficile isolates of PCR ribotype 176 for which complete genomes were generated based on hybrid assembly of Illumina and Nanopore data are highlighted with red background. Country, year of isolation, and cgMLST Finder group are shown for each strain. Circles denote acquired resistance determinants encoded by insert. Related inserts share a colour. Note that Tn6189 exists in two variants as shown in the legend. Squares show the presence of point mutations conferring resistance. A heatmap of MIC values is shown, and the colour scale is normalized for each respective breakpoint.

Figure 5.

Unrooted Maximum-Likelihood phylogenetic tree with additional RT176 C. difficile isolates. The country of origin of C. difficile isolates is colour-coded as shown in legend and the study origin is depicted by numbers in brackets: (1) isolates from this study, (2) isolates from [10], (3) isolates from [34]. A square indicates the presence of the tetM gene, while circles represent the presence of integrative conjugative elements from each family.