ClermonTyping: an easy-to-use and accurate in silico method for Escherichia genus strain phylotyping

Abstract

The genus Escherichia is composed of Escherichia albertii, E. fergusonii, five cryptic Escherichia clades and E. coli sensu stricto. Furthermore, the E. coli species can be divided into seven main phylogroups termed A, B1, B2, C, D, E and F. As specific lifestyles and/or hosts can be attributed to these species/phylogroups, their identification is meaningful for epidemiological studies. Classical phenotypic tests fail to identify non-sensu stricto E. coli as well as phylogroups. Clermont and colleagues have developed PCR assays that allow the identification of most of these species/phylogroups, the triplex/quadruplex PCR for E. coli phylogroup determination being the most popular. With the growing availability of whole genome sequences, we have developed the ClermonTyping method and its associated web-interface, the ClermonTyper, that allows a given strain sequence to be assigned to E. albertii, E. fergusonii, Escherichia clades I-V, E. coli sensu stricto as well as to the seven main E. coli phylogroups. The ClermonTyping is based on the concept of in vitro PCR assays and maintains the principles of ease of use and speed that prevailed during the development of the in vitro assays. This in silico approach shows 99.4 % concordance with the in vitro PCR assays and 98.8 % with the Mash genome-clustering tool. The very few discrepancies result from various errors occurring mainly from horizontal gene transfers or SNPs in the primers. We propose the ClermonTyper as a freely available resource to the scientific community at: http://clermontyping.iame-research.center/.

Keywords: E. coli; Escherichia; epidemiology; phylogeny; tool; typing.

Fig. 1.

ClermonTyping method flowchart. The algorithm takes a FASTA file as input and determines the species/plylogroup in two distinct ways: by Mash and in silico PCR. A warning is given when the two methods do not reach a consensus.

Fig. 2.

Complete flow scheme allowing species (Escherichia albertii, E. fergusonii, E. clades I–V and E. coli) and further E. coli phylogroup assignment used in the ClermonTyping method. The ClermonTyping method is based on the results of at least six PCR amplifications: an allele-specific amplification of chuA for E. albertii (Alb. at the top of the tree) and a specific amplification of citP for E. fergusonii (Ferg.) followed by arpA, chuA and yjaA and DNA fragment TSPE4.C2 for E. coli sensu stricto phylogroup assignment. Several additional amplifications may then be needed for a complete determination: allele-specific primers for phylogroups E and C (Gp.E and Gp.C, respectively) and allele-specific primers for Escherichia clades (Cl.I, Cl.II, Cl.III, Cl.IV and Cl.V). The amplification of trpA is used as a control for Escherichia species. The ‘Unknown’ profile will alert the user who will have to check the Mash assignation. A complete list of primers and targets is provided in Table S1.

Fig. 3.

Neighbour-joining tree depicting the phylogenetic structure of the genus Escherichia. The distances are computed by Mash on the manually curated database containing 83 strains representing the Escherichia sp./phylogroup diversity. The tree is rooted on E. albertii strains, as they are the most divergent within the genus Escherichia [1]. Bar, 0.008 Mash distance unit.

Fig. 4.

Concordance between the three methods used for Escherichia species/phylogroup assignment (PCR Clermont method in vitro, ClermonTyper and Mash). The data, presented as percentages, are based on 334 strains representing the Escherichia sp./phylogroup diversity (Table S2). The discrepancies between the PCR Clermont method in vitro and Mash are due to the limitations of the Clermont method resulting from horizontal gene transfers or SNPs in the primers. The discrepancies between the in vitro and in silico Clermont methods are due to strain contamination and IS1 insertion. Finally, concordance between the Mash and in silico Clermont methods is subject to the same bias of strain contamination and to the limitations from the original PCR Clermont method cited above.