Evolutionary and Genomic Insights into Clostridioides difficile Sequence Type 11: a Diverse Zoonotic and Antimicrobial-Resistant Lineage of Global One Health Importance

Abstract

Clostridioides difficile (Clostridium difficile) sequence type 11 (ST11) is well established in production animal populations worldwide and contributes considerably to the global burden of C. difficile infection (CDI) in humans. Increasing evidence of shared ancestry and genetic overlap of PCR ribotype 078 (RT078), the most common ST11 sublineage, between human and animal populations suggests that CDI may be a zoonosis. We performed whole-genome sequencing (WGS) on a collection of 207 ST11 and closely related ST258 isolates of human and veterinary/environmental origin, comprising 16 RTs collected from Australia, Asia, Europe, and North America. Core genome single nucleotide variant (SNV) analysis identified multiple intraspecies and interspecies clonal groups (isolates separated by ≤2 core genome SNVs) in all the major RT sublineages: 078, 126, 127, 033, and 288. Clonal groups comprised isolates spread across different states, countries, and continents, indicative of reciprocal long-range dissemination and possible zoonotic/anthroponotic transmission. Antimicrobial resistance genotypes and phenotypes varied across host species, geographic regions, and RTs and included macrolide/lincosamide resistance (Tn6194 [ermB]), tetracycline resistance (Tn6190 [tetM] and Tn6164 [tet44]), and fluoroquinolone resistance (gyrA/B mutations), as well as numerous aminoglycoside resistance cassettes. The population was defined by a large “open” pan-genome (10,378 genes), a remarkably small core genome of 2,058 genes (only 19.8% of the gene pool), and an accessory genome containing a large and diverse collection of important prophages of the Siphoviridae and Myoviridae. This study provides novel insights into strain relatedness and genetic variability of C. difficile ST11, a lineage of global One Health importance. IMPORTANCE Historically, Clostridioides difficile (Clostridium difficile) has been associated with life-threatening diarrhea in hospitalized patients. Increasing rates of C. difficile infection (CDI) in the community suggest exposure to C. difficile reservoirs outside the hospital, including animals, the environment, or food. C. difficile sequence type 11 (ST11) is known to infect/colonize livestock worldwide and comprises multiple ribotypes, many of which cause disease in humans, suggesting CDI may be a zoonosis. Using high-resolution genomics, we investigated the evolution and zoonotic potential of ST11 and a new closely related ST258 lineage sourced from diverse origins. We found multiple intra- and interspecies clonal transmission events in all ribotype sublineages. Clones were spread across multiple continents, often without any health care association, indicative of zoonotic/anthroponotic long-range dissemination in the community. ST11 possesses a massive pan-genome and numerous clinically important antimicrobial resistance elements and prophages, which likely contribute to the success of this globally disseminated lineage of One Health importance.

TheClostridioides difficilespecies problem: global phylogenomic analysis uncovers three ancient, toxigenic, genomospecies

“…The C. difficile pangenome was determined to be open (i.e. an unlimited gene repertoire) and vast in scale (over 17000 genes), much larger than previous estimates (~10000 genes) which mainly considered individual clonal lineages 16,22 . Conversely, comprising just 12.8% of its genetic repertoire (2,232 genes), the core genome of C. difficile is remarkably small, consistent with earlier WGS and microarray-based studies describing ultralow genome conservation in C. difficile 11,45 .…”

“…Genome assembly and quality control. Genomes were assembled, annotated and evaluated using a pipeline comprising TrimGalore v0.6.5, SPAdes v3.6.043, Prokka v1.14.5, and QUAST v2.344 16 . Next, Kraken2 v2.0.8-beta 60 was used to screen for contamination and assign taxonomic labels to reads and draft assemblies.…”

“…BactDating v1.0.1 62 was applied to the recombinationcorrected phylogeny produced by Gubbins (471,708 core-genome sites) with Markov chain Monte Carlo (MCMC) chains of 10 7 iterations sampled every 10 4 iterations with a 50% burn-in. A strict clock model was used with a rate of 2.5×10 -9 to 1.5×10 -8 substitutions per site per year, as previously defined by He et al 16 and Kumar et al 27 . The effective sample sizes (ESS) were >200 for all estimated parameters, and traces were inspected manually to ensure convergence.…”

“…A recombinant adjusted alignment of 471,708 polymorphic sites was used to create a core genome phylogeny with RAxML v8.2.12 (GTR gamma model of among-site rate-heterogeneity), which was visualised alongside pangenome data in Phandango 67 . Pangenome dynamics were investigated with PanGP v1.0.1 16 .…”

Global genomic population structure of Clostridioides difficile

“…Previously, genotyping was commonly performed by PCR ribotyping or DNA macrorestriction. More recent publications have documented 75 that genome-wide single-nucleotide polymorphisms (SNPs) from whole-genome sequences provide improved discrimination, and such analyses have enabled dramatic progress in our understanding of the emergence and spread of pandemic strains [9][10][11][12] and the epidemiology of local transmission 13,14 . Eyre and colleagues have argued that transmission of C. difficile isolates within a hospital environment can be 80 recognised with high probability as chains of genomes which differ by up to two SNPs whereas genomes which differ by at least 10 genomic SNPs represent unrelated bacteria 13,15 .…”