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Adaptation of host transmission cycle during Clostridium difficile speciation


Bacterial speciation is a fundamental evolutionary process characterized by diverging genotypic and phenotypic properties. However, the selective forces that affect genetic adaptations and how they relate to the biological changes that underpin the formation of a new bacterial species remain poorly understood. Here, we show that the spore-forming, healthcare-associated enteropathogen Clostridium difficile is actively undergoing speciation. Through large-scale genomic analysis of 906 strains, we demonstrate that the ongoing speciation process is linked to positive selection on core genes in the newly forming species that are involved in sporulation and the metabolism of simple dietary sugars. Functional validation shows that the new C. difficile produces spores that are more resistant and have increased sporulation and host colonization capacity when glucose or fructose is available for metabolism. Thus, we report the formation of an emerging C. difficile species, selected for metabolizing simple dietary sugars and producing high levels of resistant spores, that is adapted for healthcare-mediated transmission.

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Fig. 1: Phylogeny and population structure of Clostridium difficile.
Fig. 2: Adaptation of sporulation and metabolic genes in Clostridium difficile clade A.
Fig. 3: Bacterial speciation is linked to increased host adaptation and transmission ability.

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Data availability

Genomes have been deposited in the European Nucleotide Archive. Accession codes are listed in Supplementary Table 1. The 13 C. difficile reference isolates (Supplementary Table 2) are publicly available from the NCTC and the annotation of these genomes are available from the Host-Microbiota Interactions Laboratory (HMIL;, Wellcome Sanger Institute.

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This work was supported by the Wellcome Trust (098051), the UK Medical Research Council (PF451 and MR/K000511/1), the Australian National Health and Medical Research Council (1091097 and 1159239 to S.F.) and the Victorian Government’s Operational Infrastructure Support Program. The authors thank S. Weese, F. Miyajima, G. Songer, T. Louie, J. Rood and N. M. Brown for C. difficile strains. The authors thank A. Neville, D. Knight and B. Hornung for critical reading and comments. The authors would also like to acknowledge the support of the Wellcome Sanger Institute Pathogen Informatics Team.

Author information

Authors and Affiliations



N.K. and T.D.L. conceived and managed the study. N.K., S.C.F., E.V., H.P.B. and T.D.L. wrote the manuscript. D.J.F., P.R., M.P., M.RJ.C., M.B.F.J., K.R.H., M.I., L.H.W., C.S., T.N., G.D., T.V.R., E.J.K. and B.W.W. provided critical input and contributed to the editing of the manuscript. N.K. performed the computational analysis. H.P.B. performed genome annotation of reference genomes. D.J.F., P.R., M.P., M.RJ.C., M.B.F.J., K.R.H., M.I., L.H.W., C.S. and T.N. obtained C. difficile strains. E.V., H.P.B., S.C.F. and T.D.L. designed in vitro and in vivo experiments. H.P.B., E.V. and M.S. performed in vitro experiments. E.V., M.D.S., S.C. and K.H. performed in vivo experiments.

Corresponding authors

Correspondence to Nitin Kumar or Trevor D. Lawley.

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The authors declare no competing interests.

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Integrated supplementary information

Supplementary Figure 1 Breakdown of 906 Clostridium difficile strains based on metadata.

(a) Number of strains based on geographical location is shown in bar-plots. (b) Number of strains based on source.

Supplementary Figure 2 Pairwise SNPs difference between different phylogenetic groups of Clostridium difficile.

Boxplots show distribution of SNPs differences calculated between pairs of genomes belonging to different PGs (PG1: n = 108 genomes, PG2: n = 398 genomes, PG3: n = 112 genomes, PG4: n = 288 genomes). Box plots show minimum to maximum values and the median value.

Supplementary Figure 3 Colony morphology of Clostridium difficile strains.

C. difficile strains from distinct clades were plated on YCFA agar plates supplemented with 0.1% sodium taurocholate and incubated for 8 days and C. difficile colonies were photographed. Ribotype RT002, RT027, and RT017 represent PG1, 2 and 3 respectively. RT045, RT078 and RT033 represent PG4. Experiment was repeated 3 time with similar results.

Supplementary Figure 4 Bayesian skyline plots.

Skyline plot of Clostridium difficile PG2 (RT027; n = 44 strains) and PG4 (RT078; n = 97 strains) indicate signals of C. difficile clade A expansion in the year 1595. The black line represents median estimate, and purple area represents its 95% highest posterior density intervals.

Supplementary Figure 5 Recombination analysis based on whole genome of 906 Clostridium difficile strains.

Phylogenetic groups of C. difficile are shown in circles. Direction of edges represent direction of recombination event (donor to recipient). Range of recombination events are shown on the edges.

Supplementary Figure 6 Comparison of accessory genome between 4 phylogenetic groups (PGs) of Clostridium difficile.

(a) Discriminant analysis of principal components using Clusters of Orthologous Groups (COGs) and accessory genome of strains from PG1 (n = 108 genomes), PG2 (n = 398 genomes), PG3 (n = 112 genomes), and PG4 (n = 288 genomes). (b) Functional classification and distribution of enriched genes in the group of PG1, 2 and 3 (n = 618 genomes) as compared to PG4 (n = 288 genomes). Cell motility (including flagella) and mobile elements are the most enriched functions. (c) Functional classification and distribution of enriched genes in PG4 (n = 288 genomes) as compared to the group of PG1, 2 and 3 (n = 618 genomes). Uncharacterized functions and DNA replication and modification functions are the most enriched functions. One-sided Fisher’s exact test with p-value adjusted by Hochberg method.

Supplementary Figure 7 High number of pseudogenes in the Clostridium difficile clade A compared to clade B.

The bar-plot shows the number of pseudogenes in each phylogenetic group (PG1: n = 108 genomes, PG2: n = 398 genomes, PG3: n = 112 genomes, PG4: n = 288 genomes).

Supplementary Figure 8 Sporulation-associated genes in Clostridium difficile clade B.

There are 21 sporulation-associated positively selected genes in PG4. These are all either present in the mature spore proteome or they are regulated by Spo0A or its sporulation specific sigma factors. There are no genes directly involved in producing a spore in any of the sporulation stages.

Supplementary Figure 9 Multiple sequence alignment of the sodA gene from Clostridium difficile clade A and clade B.

A nucleotide consensus sequence for 4 phylogenetic groups (PG1-4) is shown. Three-point mutations which are present in all C. difficile clade A genomes and absent in C. difficile clade B genomes are shown in black boxes. The amino-acids related to these mutations are mentioned.

Supplementary Figure 10 Schematic diagram showing the metabolic pathway of glucose and fructose metabolism in C. difficile.

Positively selected genes of Clostridium difficile clade A are shown in blue.

Supplementary Figure 11 Functional diversity of carbohydrate-active enzyme in 4 phylogenetic groups (PGs) of Clostridium difficile.

Discriminant analysis of principal components using carbohydrate active enzymes (CAZymes) database. Each color represents a strain from 4 PGs: PG1 (n = 108 genomes); PG2 (n = 398 genomes); PG3 (n = 112 genomes) and PG4 (n = 288 genomes). One-sided Fisher’s exact test with p-value adjusted by Hochberg method.

Supplementary information

Supplementary Information

Supplementary Figs. 1–11

Reporting Summary

Supplementary Table 1

List of Clostridium difficile strains included in this study.

Supplementary Table 2

List of high-quality genomes of Clostridium difficile strains.

Supplementary Table 3

List of 1322 single copy core genes present in 906 Clostridium difficile strains.

Supplementary Table 4

List of accessory genes enriched in Clostridium difficile clade A (n = 618 genomes). One-sided Fisher’s exact test with p-value adjusted by Hochberg method.

Supplementary Table 5

List of accessory genes enriched in Clostridium difficile clade B (n = 288 genomes). One-sided Fisher’s exact test with p-value adjusted by Hochberg method.

Supplementary Table 6

List of pseudogenes in Clostridium difficile PG1

Supplementary Table 7

List of pseudogenes in Clostridium difficile PG2.

Supplementary Table 8

List of pseudogenes in Clostridium difficile PG3.

Supplementary Table 9

List of pseudogenes in Clostridium difficile PG4.

Supplementary Table 10

List of pseudogenes that are present in all phylogenetic groups of Clade A but absent in Clade B of Clostridium difficile.

Supplementary Table 11

List of pseudogenes that are present in Clade B but absent in Clade A of Clostridium difficile.

Supplementary Table 12

List of positively selected genes in Clostridium difficile clade A

Supplementary Table 13

List of positively selected genes in Clostridium difficile clade B.

Supplementary Table 14

Presence/absence matrix of carbohydrate-active enzyme in 4 phylogenetic groups of Clostridium difficile.

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Kumar, N., Browne, H.P., Viciani, E. et al. Adaptation of host transmission cycle during Clostridium difficile speciation. Nat Genet 51, 1315–1320 (2019).

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