Distribution of Clostridioides difficile ribotypes and sequence types across humans, animals and food in 13 European countries

Abstract

Clostridioides difficile is a One Health pathogen found in humans, animals, and the environment, with food representing a potential transmission route. One Health studies are often limited to a single country or selected reservoirs and ribotypes. This study provides a varied and accessible collection of C. difficile isolates and sequencing data derived from human, animal, and food sources across 13 European countries. A total of 441 strains (human hospital- and community-associated cases n = 280, animal n = 96, food n = 65) were analysed by ribotyping, toxinotyping and whole-genome sequencing (WGS). We detected 83 sequence types (STs), with ST11 (n = 80 isolates) and ST1 (n = 54 isolates) being the most represented. Several STs included strains originating from all source combinations. Further genomic analysis confirmed close genetic relatedness in some of the STs. Additionally, the genomic analysis identified 10 strains from cryptic clades (C-I to C-III) and 4 of them were mono-toxigenic possessing only a variant form of tcdA gene. Amongst 106 ribotypes, 10 were shared between all 3 sources and 68 were source-specific. Some ribotypes were only found at the intersection of human and food source (RT023, RT027), or between human and animal source (RT009, RT045, RT046). C. difficile ribotypes and STs in Europe were diverse. In this collection, some ribotypes showed potential association with food or animal transmission routes. C. difficile strains from divergent clades CI-III, currently emerging in the human population, were rare and mostly food-associated.Trial registration: ClinicalTrials.gov identifier: NCT03503474.

Keywords: One Health; animals; community-associated; cryptic clades; epidemiology; food; hospital-associated; transmission.

Figure 1.

COMBACTE-CDI sequenced isolates analysed by whole-genome MLST (wgMLST) in EPISEQ®CS application (n = 430). The Minimum Spanning Tree (MST) shows the relationships between all 430 isolates based on their wgMLST profiles. The MST displayed within the EPISEQ®CS application was further edited as described in methods. Each node (circle) corresponds to one isolate and are coloured according to host source. Distances in tree (allele differences) are drawn to scale, with values of 250 or more between two isolates shown next to the branches connecting them. All distances, including those less than 250 are provided in Supplementary Table S3. The sequence types (STs) calculated by EPISEQ®CS (Oxford MLST scheme) are shown next to each node or clusters of nodes, with corresponding ribotypes (RTs) in brackets. STs or RTs identified in at least 1% of the 430 isolates are shown in bold. Most prevalent STs (>1%) are highlighted in a grey or coloured (top five) shadowing. Additional metadata (isolate ID, country of origin, toxinotyping) is provided in the wgMLST-based phylogenetic analysis dendrogram (Supplementary Figure S1).

Figure 2.

Selected clusters from overall minimum spanning tree of all COMBACTE-CDI sequenced isolates. Each dot represents one isolate. EPISEQ®CS wgMLST-based allelic differences are shown in branches between isolates. The source and ribotype of each isolates are colour coded.

Figure 3.

wgSNP-based neighbour-joining phylogenetic tree of RT027, RT045, RT078 and RT126 isolates with close genetic relatedness. Strains in RT045, RT078 and RT126 have less than 10 SNPs and are closely related. Isolates are named with GSK and number, followed by source and country.

Figure 4.

Distribution of C. difficile PCR ribotypes across four different sources. Strains (n = 441) were isolated in 13 EU countries, are primarily contemporary strains from the year 2018 and include human hospital strains, human community strains, animal strains (mostly pigs), food strains (potato). (A) Venn diagram showing shared and source-specific PCR ribotypes. (B) Most common PCR ribotypes for each source ordered by number of strains; *non-toxigenic strain.

Figure 5.

Distribution of most common C. difficile PCR ribotypes in the strain collection originating from 2018 from representative European countries included in COMBACTE-CDI study. Strains were obtained from humans (hospital or community source), animals (mainly pigs but also other species) and from retail potato samples. While there is unequal distribution of isolates of different sources from different countries, the COMBACTE-CDI strain collection still represents well the overview of main PCR ribotypes found in each source (listed in the legends). Pie chart diagrams show the distribution for the individual country; number in the circle is the number of analysed isolates.