Determining Clostridium difficile intra-taxa diversity by mining multilocus sequence typing databases

Abstract

Background: Multilocus sequence typing (MLST) is a highly discriminatory typing strategy; it is reproducible and scalable. There is a MLST scheme for Clostridium difficile (CD), a gram positive bacillus causing different pathologies of the gastrointestinal tract. This work was aimed at describing the frequency of sequence types (STs) and Clades (C) reported and evalute the intra-taxa diversity in the CD MLST database (CD-MLST-db) using an MLSA approach.

Results: Analysis of 1778 available isolates showed that clade 1 (C1) was the most frequent worldwide (57.7%), followed by C2 (29.1%). Regarding sequence types (STs), it was found that ST-1, belonging to C2, was the most frequent. The isolates analysed came from 17 countries, mostly from the United Kingdom (UK) (1541 STs, 87.0%). The diversity of the seven housekeeping genes in the MLST scheme was evaluated, and alleles from the profiles (STs), for identifying CD population structure. It was found that adk and atpA are conserved genes allowing a limited amount of clusters to be discriminated; however, different genes such as drx, glyA and particularly sodA showed high diversity indexes and grouped CD populations in many clusters, suggesting that these genes' contribution to CD typing should be revised. It was identified that CD STs reported to date have a mostly clonal population structure with foreseen events of recombination; however, one group of STs was not assigned to a clade being highly different containing at least nine well-supported clusters, suggesting a greater amount of clades for CD.

Conclusions: This study shows the usefulness of CD-MLST-db as a tool for studying CD distribution and population structure, identifying the need for reviewing the usefulness of sodA as housekeeping gene within the MLST scheme and suggesting the existence of a greater amount of CD clades. The study also shows the plausible exchange of genetic material between STs, contributing towards intra-taxa genetic diversity.

Keywords: Clostridium difficile; Distribution pattern; Multilocus sequence typing (MLST); Population structure.

Fig. 1

Epidemiological profiles of CD isolates reported in CD-MLST-db. a Frequency of clades to which all the STs reported belonged. b Frequency of main STs reported (only clades having ≥ 1.0% frequency are shown). c Geographical distribution of CD clades by country of origin. d. Temporal distribution (per year) by clade of reported STs. Percentages were calculated regarding all isolates included in the analysis (n: 1,772). The bottom map used was retrieved from https://www.google.com/about/datacenters/inside/locations/index.html

Fig. 2

Graphic representation of nucleotide diversity (π) by: a housekeeping gene; b country and c clade

Fig. 3

Phylogenetic analysis from concatenated sequences of the total set of STs (n: 380). The final tree was obtained with Neighbour Joining method using MEGA 7. The red arrows denote the STs with incongruence in their clustering. Black bars mark the STs that are not grouped in a single Clade/Cluster

Fig. 4

Allelic plot. Graphic representation of phylogenetic inferences where a colour is assigned to each cluster (Bootstrap ≥80.0%) found per gene (row). For each ST (columns) the cluster to which it belongs was analysed. The first row describes traditional classification per clade for CD. The upper section shows the results for all STs (n: 380) and the lower section shows the zoom of the STs defined as NV

Fig. 5

Phylogenetic reconstruction from concatenated sequences of STs grouped as NV. Well-supported clusters are highlighted in grey (n: 10 cluster) and black dots represent bootstrap values ≥ 80.0%