Genomic epidemiology of Clostridioides difficile sequence type 35 reveals intraspecies and interspecies clonal transmission

Abstract

Clostridioides difficile sequence type (ST) 35 has been found in humans and animals worldwide. However, its genomic epidemiology and clonal transmission have not been explored in detail. In this study, 176 C. difficile ST35 isolates from six countries were sequenced. Genomic diversity, clonal transmission and epidemiological data were analyzed. Sporulation and virulence capacities were measured. Four ribotypes (RT) were identified including RT046 (97.2%), RT656 (1.1%), RT427 (0.6%), and RT AI-78 (1.1%). Phylogenetic analysis of 176 ST35 genomes, along with 50 publicly available genomes, revealed two distinctive lineages without time-, region-, or source-dependent distribution. However, the distribution of antimicrobial resistance genes differed significantly between the two lineages. Nosocomial and communal transmission occurred in humans with the isolates differed by ≤ two core-genome single-nucleotide polymorphism (cgSNPs) and clonal circulation was found in pigs with the isolates differed by ≤ four cgSNPs. Notably, interspecies clonal transmission was identified among three patients with community acquired C. difficile infection and pigs with epidemiological links, differed by ≤ nine cgSNPs. Toxin B (TcdB) concentrations were significantly higher in human isolates compared to pig isolates, and ST35 isolates exhibited stronger sporulation capacities than other STs. Our study provided new genomic insights and epidemiological evidence of C. difficile ST35 intraspecies and interspecies clonal transmission, which can also be facilitated by its strong sporulation capacity.

Keywords: Clostridioides difficile; ST35; clonal transmission; genome epidemiology; sporulation capacity; virulence; whole genome sequencing.

Figure 1.

Dendrogram of seven ribotyping patterns by capillary gel electrophoresis. The dendrograms were clustered using UPGMA. The length of branch was labelled above each branch.

Figure 2.

Maximum likelihood tree of 226 C. difficile ST35 genomes. The bootstraps were marked on each branch. Lineage 1 (L1) and lineage 2 (L2) were marked with blue and red full lines. Two main clusters (C1 and C2) were collapsed to reduce figure size.

Figure 3.

Genetic relationships among C. difficile ST35 isolates determined by cgSNP analysis. A: geographical locations of farms and hospitals; B: timelines of C. difficile ST35 isolates from four hospitals and different farms. Patients with CDI on the same blue line were admitted to the same hospital, pigs with different colors indicated different farms. The red numbers represented six patients from the same department. The light orange, purple, and blue frames illustrated the difference in cgSNPs between the isolates; C: the numbers of cgSNP difference were shown among nine isolates including three human and six pig isolates.

Figure 4.

Comparison of TcdB production and determination of sporulation among the different groups. A: TcdB concentrations measured using the real-time cell analysis; B: sporulation capacity measured using the heat-induced method. **represented significant difference (P < 0.01). ZJ: Zhejiang, HB: Hebei, SH: Shanghai.