Genomic study of European Clostridioides difficile ribotype 002/sequence type 8

Abstract

Clostridioides difficile has significant clinical importance as a leading cause of healthcare-associated infections, with symptoms ranging from mild diarrhoea to severe colitis, and possible life-threatening complications. C. difficile ribotype (RT) 002, mainly associated with MLST sequence type (ST) 8, is one of the most common RTs found in humans. This study aimed at investigating the genetic characteristics of 537 C. difficile genomes of ST8/RT002. To this end, we sequenced 298 C. difficile strains representing a new European genome collection, with strains from Germany, Denmark, France and Portugal. These sequences were analysed against a global dataset consisting of 1,437 ST8 genomes available through Enterobase. Our results showed close genetic relatedness among the studied ST8 genomes, a diverse array of antimicrobial resistance (AMR) genes and the presence of multiple mobile elements. Notably, the pangenome analysis revealed an open genomic structure. ST8 shows relatively low overall variation. Thus, clonal isolates were found across different One Health sectors (humans, animals, environment and food), time periods, and geographical locations, suggesting the lineage's stability and a universal environmental source. Importantly, this stability did not hinder the acquisition of AMR genes, emphasizing the adaptability of this bacterium to different selective pressures. Although only 2.4 % (41/1,735) of the studied genomes originated from non-human sources, such as animals, food, or the environment, we identified 9 cross-sectoral core genome multilocus sequence typing (cgMLST) clusters. Our study highlights the importance of ST8 as a prominent lineage of C. difficile with critical implications in the context of One Health. In addition, these findings strongly support the need for continued surveillance and investigation of non-human samples to gain a more comprehensive understanding of the epidemiology of C. difficile.

Keywords: Clostridioides difficile; One Health; RT002; ST8; genomes; pangenome; phylogenetic analysis; zoonotic pathogen.

Fig. 1.. Phylogenetic tree (cgSNP) of C. difficile ST8/RT002. In this tree, 298 FED-AMR genomes, 239 dereplicated (see Methods section, ‘ST8/RT002 genome collection’) Enterobase genomes and one reference genome (W0003a, accession number NZ_CP025047) were included. There is no obvious clustering of antimicrobial resistance determinants or transposons in special branches of the tree, and neither is there clustering according to the geographical origin or the sample type to special branches. It is striking that 73 % (19/26) of Asian genomes harbour antimicrobial resistance determinants. The phylogenetic tree was created with iTOL (v. 6.6) [37]. A web version of the tree can be accessed at https://itol.embl.de/tree/193221152140881717331704.

Fig. 2.. Pangenome and core genome of C. difficile ST8/RT002. The pangenome analysis was conducted with the software panaroo. (a) The total number of genes of the pangenome (blue) and the core genome (green) of C. difficile ST8/RT002 were calculated. The pangenome of 538 investigated genomes comprised 6,173 genes. (b) The number of new gene clusters per sequentially added genome are depicted as green columns.

Fig. 3.. Antimicrobial resistance profiles of selected resistance determinants in ST8/RT002 per continent. All detected antimicrobial resistance (AMR) determinants are included except for those commonly found in ST8/RT002 [cdeA, blaR1, bla_CDD-1, bla_CDD-2, van gene cluster (vanG, vanRC, vanSC and vanTC), vanZA and abc-f (vga(D)-related)]. The number of investigated genomes can be found in parentheses after the respective continent. AMR determinants were detected in 73.1 % (19/26) of the Asian ST8/RT002 genomes, conferring resistance to fluoroquinolones, macrolides, tetracycline and aminoglycosides, while 4.1 % (17/411) of the European and 6.7 % (4/60) of the North American genomes harboured AMR determinants.

Fig. 4.. Phylogenetic tree of ST8/RT002 (cgSNP) with detected plasmids and prophages. In this tree, 298 FED-AMR genomes, 239 dereplicated (see Methods section, ‘ST8/RT002 genome collection’) Enterobase genomes and one reference genome were included. This phylogenetic tree was created according to results of the cgSNP analysis. It shows the occurrence of plasmids (purple) and prophages (blue) for each genome. The phylogenetic tree was created with iTOL (v. 6.6) [37].

Fig. 5.. cgMLST ST8 clusters (threshold of three differing alleles) that contain C. difficile genomes derived from human and non-human samples. Only genomes with a given collection year are included in the figure. AU, Australia; BE, Belgium; CA, Canada; DE, Germany; DK, Denmark; NL, The Netherlands; PL, Poland; PT, Portugal; SI, Slovenia; UK, United Kingdom, US, United States of America.