Genome Analysis of Clostridium difficile PCR Ribotype 014 Lineage in Australian Pigs and Humans Reveals a Diverse Genetic Repertoire and Signatures of Long-Range Interspecies Transmission

Abstract

Clostridium difficile PCR ribotype (RT) 014 is well-established in both human and porcine populations in Australia, raising the possibility that C. difficile infection (CDI) may have a zoonotic or foodborne etiology. Here, whole genome sequencing and high-resolution core genome phylogenetics were performed on a contemporaneous collection of 40 Australian RT014 isolates of human and porcine origin. Phylogenies based on MLST (7 loci, STs 2, 13, and 49) and core orthologous genes (1260 loci) showed clustering of human and porcine strains indicative of very recent shared ancestry. Core genome single nucleotide variant (SNV) analysis found 42% of human strains showed a clonal relationship (separated by ≤2 SNVs in their core genome) with one or more porcine strains, consistent with recent inter-host transmission. Clones were spread over a vast geographic area with 50% of the human cases occurring without recent healthcare exposure. These findings suggest a persistent community reservoir with long-range dissemination, potentially due to agricultural recycling of piggery effluent. We also provide the first pan-genome analysis for this lineage, characterizing its resistome, prophage content, and in silico virulence potential. The RT014 is defined by a large "open" pan-genome (7587 genes) comprising a core genome of 2296 genes (30.3% of the total gene repertoire) and an accessory genome of 5291 genes. Antimicrobial resistance genotypes and phenotypes varied across host populations and ST lineages and were characterized by resistance to tetracycline [tetM, tetA(P), tetB(P) and tetW], clindamycin/erythromycin (ermB), and aminoglycosides (aph3-III-Sat4A-ant6-Ia). Resistance was mediated by clinically important mobile genetic elements, most notably Tn6194 (harboring ermB) and a novel variant of Tn5397 (harboring tetM). Numerous clinically important prophages (Siphoviridae and Myoviridae) were identified as well as an uncommon accessory gene regulator locus (agr3). Conservation in the pathogenicity locus and S-layer correlated with ST affiliation, further extending the concept of clonal C. difficile lineages. This study provides novel insights on the genetic variability and strain relatedness of C. difficile RT014, a lineage of emerging One Health importance. Ongoing molecular and genomic surveillance of strains in humans, animals, food, and the environment is imperative to identify opportunities to reduce the overall CDI burden.

Keywords: CDI; One Health; antimicrobial resistance; pan-genome; phylogenomics; porcine; zoonosis.

Figure 1

MLST, antimicrobial resistance, and prophage analysis for C. difficile RT014 from humans and animals in Australia. (A) Maximum likelihood MLST phylogeny. Scale shows the number of substitutions per site, based on concatenated MLST allele sequences (7 loci, 3501 bp). For global phylogenetic context, well-characterized representatives of MLST clades 1 (ST54, RT012), 2 (ST1, RT027), 3 (ST22, RT023), 4 (ST37, RT017), and 5 (ST11, RT078) are also shown (^*). For comparative purposes ST14 (RT020), an RT often grouped with RT014 is also included. Tree is mid-point rooted and is supported by 1000 bootstrap replicates (only values >50 are shown). The branch depicting divergent ST 11 contains a break; the overall length of this branch is 0.0144. Branch and taxa coloring/labeling for RT014 strains; teal, human (H); purple, porcine (P); red, ST2 (n = 21); green, ST13 (n = 16); blue, ST49 (n = 7). (B) Heatmap visualizing the distribution of antimicrobial resistance elements, associated phenotypes and prophage content. Presence (), absence (), MICs were not determined for UK strains Ox1533, Ox1475, Ox1593, and ATCC43600 (). Some genomes harbored duplicate copies of prophages; P3 (2x ΦC2), P7 (2x ΦC2), P15 (3x ΦC2), H8 (2x ΦC2), H19 (2x ΦC2 and 2x ΦMMP02), and Ox1475 (2x ΦC2).

Figure 2

Population structure of 44 C. difficile RT014 based on core orthologous genes. ClonalframeML radial phylogram constructed from gene-by-gene alignment of 1260 orthologous gene clusters (1,019,160 bp). Tree is mid-point rooted and the nodes are supported by 1000 bootstrap replicates (only values >50 are shown). Scale bar represents the number of the number of substitutions per nucleotide site. Branch and taxa coloring/labeling for RT014 strains; teal, human (H); purple, porcine (P); red, ST2 (n = 21); green, ST13 (n = 16); blue, ST49 (n = 7). Boxes (A,B) indicate interspecies clustering in ST lineages 2 and 13, respectively. Legend shows corresponding information for strain ID, origin, site, and date of collection.

Figure 3

Single nucleotide variant analysis of 44 C. difficile RT014. Maximum-likelihood phylogeny based on non-recombinant SNVs (n = 1287) identified after mapping all sequence reads against the CD630 reference genome (accession AM180355, 4,290,252 bp). RAxML tree is mid-point rooted and is supported by 1000 non-parametric bootstrap replicates (only values >50 are shown). Branch and taxa coloring/labeling for RT014 strains; teal, human (H); purple, porcine (P); red, ST2 (n = 21); green, ST13 (n = 16); blue, ST49 (n = 7). Taxa labels include ID: ORIGIN-SITE, ISOLATION DATE, and ACQUISITION STATUS (if known). The black boxes indicate a clonal group (CG) where all isolates differ by no more than two SNVs (0–2). To enhance the visual resolution of the relative evolutionary distances (branch lengths/tips) between test genomes, CD630 was omitted from the final phylogeny (mean 1069 SNV differences from test genomes).

Figure 4

Core genome SNV distances between 44 C. difficile RT014. Heatmap of pairwise core genome SNV differences (Δ) between all 44 isolates, sorted by ST: red, ST2 (n = 21); green, ST13 (n = 16); and blue, ST49 (n = 7).

Figure 5

Genetic organization of Tn5397 in RT014 genomes. A representative Tn5397-like element from porcine strain P5 is shown compared to conjugative transposon Tn5397 (AF333235.1). Arrows indicate open reading frames (ORFs) and direction of transcription. Characteristic features tetM (tetracycline resistance gene), tndX (site-specific recombinase gene) and ORF 14 are shown in brown, orange and pink respectively, with the remaining ORFs shown in green. ORF 14 is significantly truncated in P5 and completely lacks the 1831 bp group II intron (ORF 14^*), a characteristic feature of Tn5397. Figure prepared using Easyfig (Sullivan et al., 2011). Gray vertical blocks between sequences indicate regions of homology (Blast nucleotide identity, >99%). Overall size (and GC%) of elements in P5 and AF333235.1 are 18,011 bp (38.5%) and 20,658 bp (38.4%), respectively.

Figure 6

Core and pan-genome analysis for 44 C. difficile RT014 genomes. The total number of genes in the pan (blue) and core (green) genomes are plotted as a function of the number of genomes sequentially added (n = 44). Box plots indicate 25th and 75th percentiles with medians shown as horizontal lines and whiskers set at 10th and 90th percentiles. For the pan-genome, the continuous curve (blue) shows the fit (r² = 0.999) of the power-law regression model. The pan-genome size is calculated at 7587 genes at n = 44 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) Bpan (≈ γ, Tettelin et al., 2008) was estimated as 0.43. For the core genome, the continuous curve (green) shows the fit (r² = 0.979) of the exponential regression model. The number of core genes converges to 2296 at n = 44, accounting for 30.3% of the total gene repertoire.

Figure 7

Strain-specific gene analysis for 44 C. difficile RT014 genomes. A plot of the number of new “strain-specific” genes contributing to the gene pool per additional sequenced strain as a function of the number of strains (n = 44). Consistent with an open pan-genome, the number of new genes does not converge to zero upon sequencing of additional genomes (at n = 44, an average of 48 new genes are contributed to the gene pool).

Figure 8

Proteome analysis for 44 C. difficile RT014 genomes. Functional proteomic characterization of CDS identified in the C. difficile RT014 pan-genome (n = 44). A total of 3355 CDS (44.22%) were classified by KEGG. CDS categorized as “unclassified” were identified by but no functional classification currently exists.