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.

Keywords: Clostridium difficile; One Health; antimicrobial resistance; epidemiology; evolution; livestock; microbial genomics; toxin; transmission; zoonosis.

FIG 1

Molecular epidemiology. PCR ribotyping banding patterns for 16 unique C. difficile RTs analyzed in this study (n = 207). The corresponding toxin gene profile is also provided. Origin: CLIN, clinical; VET, veterinary; ENV, environmental; RT, PCR ribotype; QX, novel RT assignment (internal nomenclature). Superscript a indicates the molecular epidemiology for some of these strains is based on previously published works (4, 36).

FIG 2

Microevolutionary analysis and clonal transmission. (A) Maximum likelihood phylogeny of 207 C. difficile genomes based on evolution in 1,076 nonrecombinant, nonrepetitive core genome SNVs in clonal frame. Taxa are colored according to RT lineage: RT033/288 (green; n = 33), RT078 (red; n = 40), RT126 (blue; n = 69), RT127 (orange; n = 54), or other (gray; n = 11). Strain origin is indicated in yellow (clinical, taxa prefixed with “C”) and purple (veterinary/environmental, taxa prefixed with “V/E”). Clonal relationships (two or more strains sharing ≤2 core genome SNVs) are indicated in black. The tree is midpoint rooted, and the nodes are supported by 1,000 nonparametric bootstrap replicates (values of >95 are shown [*]). The overall topology supports PCR ribotype assignment with six major strain clusters identified (the RT126/078 cluster, RT127 clusters I to III, the RT033/288 cluster, and the sequence type 258 [ST258] cluster). (B) Distribution plots showing core genome SNV distances between each strain and the genetically closest strain in each cluster. Vertical lines represent the 2-SNV cutoff for the identification of clonally transmitted strains (18). In “RT127*,” the asterisk indicates that clusters I to III are merged.

FIG 3

In vitro antimicrobial susceptibility. (A) MIC distributions for 13 antimicrobial agents against 185 C. difficile. VAN, vancomycin; MTZ, metronidazole; FDX, fidaxomicin; RFX, rifaximin; AMC, amoxicillin-clavulanate; CLI, clindamycin; ERY, erythromycin; CRO, ceftriaxone; MEM, meropenem; MXF, moxifloxacin; TET, tetracycline; TZP, piperacillin-tazobactam; TMP, trimethoprim. Where available, established susceptible and resistant breakpoints are indicated by vertical green and red lines, respectively. (B) Plots showing overall percentage of nonsusceptibility and percentage of phenotypic-genotypic concordance for the TetR (tetracycline-resistant), FQR (fluoroquinolone-resistant), and MLS_B (macrolide-lincosamide-streptogramin B-resistant) phenotypes.

FIG 4

Comparative analysis of antimicrobial resistance (AMR) and virulence loci. Shown are heat maps visualizing the distribution of AMR (A) and virulence/pathogenicity (B) loci across the 207-genome data set. Presence is indicated by black bars and absence by light blue bars. Strains are arranged from left to right according to RT lineage: RT033/288 (green; n = 33), RT078 (red; n = 40), RT126 (blue; n = 69), RT127 (orange; n = 54), and other (gray; n = 11). Strain origin is also indicated in yellow (clinical) and purple (veterinary/environmental). Superscript a indicates results comprising syntenic vanXB, vanB, vanHB, vanW, vanYB, vanSB, and vanRB genes. Superscript b indicates the combination of QRDR mutations in gyrA (Thr82Ile) and gyrB (Ser366Val, Ser416la, Asp426Asn, and Glu466Val). Superscript c indicates results are according to the scheme of Dingle et al. (21). (Alleles 20 and 21 are novel and were identified in this study.) Superscript d indicates results are according to the scheme of Curry et al. (23) (characterized by C→T substitution at nucleotide 184 and an in-frame deletion of 39 bp at nucleotide positions 341 to 379). Superscript e indicates the wild-type (WT) 747-bp cdtR allele. Superscript f indicates the variant (VNT) 324-bp cdtR allele. Superscript g indicates results are according to the scheme of Dingle et al. (21) (characterized by diversity in slpA, cwp66, cd2790, cwp2, and secA2). (Cassette type DK1 is novel and was identified in this study but has yet to be assigned an official number by the curators of the Bacterial Isolate Genome Sequence Database.)

FIG 5

tcdB receptor binding domain (RBD) diversity. (A) Organization of the four functional domains of the 2,366-amino-acid TcdB protein. The 597-bp variable region within the C-terminus receptor binding domain (RBD) is indicated (horizontal red bar). The figure was adapted from Dingle et al. (21). (B) Neighbor-joining phylogeny for 21 currently described tcdB RBD alleles. Tips are colored according to the MLST clade (see the color key). Novel alleles identified in this study (open green circles) are clustered with known MLST clade 5 allele type 1 (reported to date in the RT078 and RT126 lineages). Each sequence is 198 amino acid residues in length. Sequences were aligned using MUSCLE, and the tree was generated in MEGA6 with evolutionary distances calculated using the Tajima-Nei model. The scale bar shows the number of amino acid substitutions per site. The tree is midpoint rooted and supported by 500 bootstrap replicates. (C) Summary of nucleotide and amino acid changes in novel tcdB RBD variants.

FIG 6

Pan-genome and prophage content. The total numbers of genes in the pan-genome (A) and core genome (B) are plotted as a function of the number of genomes sequentially added (n = 207). (A) The pan-genome size is calculated at 10,378 genes at n = 207 and displays characteristics of an open genome: (i) the trajectory of the pan-genome increases unboundedly as the number of genomes are added, and (ii) B_pan (≈γ [55]) was estimated as 0.46 (curve fit, r² = 0.999). Box plots indicate the 25th and 75th percentiles, with medians shown as horizontal lines and whiskers set at the 10th and 90th percentiles. (B) Consistent with an open pan-genome, the core genome curve (r² = 0.985) converges to 2,058 genes at n = 207, where an average of 16 new strain-specific genes are contributed to the gene pool. Overall, the core genome accounts for just 19.8% of the total gene repertoire. (C) Summary of intact prophage content found in 207 C. difficile strains of ST11 and ST258. More prophages were found in LCT⁻ RTs versus LCT⁺ RTs, the RT127 lineage versus the RT126 and -078 lineages, and veterinary versus clinical strains (P < 0.001).