Predictive fluorescent amplified-fragment length polymorphism analysis of Escherichia coli: high-resolution typing method with phylogenetic significance

Abstract

The fluorescent amplified-fragment length polymorphism (FAFLP) assay potentially amplifies a unique set of genome fragments from each bacterial clone. It uses stringently hybridizing primers which carry a fluorescent label. Precise fragment sizing is achieved by the inclusion of an internal size standard in every lane. Therefore, a unique genotype identifier(s) can be found in the form of fragments of precise size or sizes, and these can be generated reproducibly. In order to evaluate the potential of FAFLP as an epidemiological typing method with a valid phylogenetic basis, we applied it to 87 strains of Escherichia coli. These comprised the EcoR collection, which has previously been classified by multilocus enzyme electrophoresis (MLEE) and which represents the genetic diversity of the species E. coli, plus 15 strains of the clinically important serogroup O157. FAFLP with an unlabelled nonselective EcoRI primer (Eco+0) and a labelled selective MseI primer (Mse+TA) gave strain-specific profiles. Fragments of identical sizes (in base pairs) were assumed to be identical, and the genetic distances between the strains were calculated. A phylogenetic tree derived from measure of distance correlated closely with the MLEE groupings of the EcoR collection and placed the verocytotoxin-producing O157 strains on an outlier branch. Our data indicate that FAFLP is suitable for epidemiological investigation of E. coli infection, providing well-defined and reproducible identifiers of genotype for each strain. Since FAFLP objectively samples the whole genome, each strain or isolate can be assigned a place within the broad context of the whole species and can also be subjected to a high-resolution comparison with closely related strains to investigate epidemiological clonality.

FIG. 1

Sizes (in base pairs) and approximate locations of FAFLP assay-predicted fragments in the E. coli K-12 MG1655 genome obtained with primer set MseI+TA–EcoRI+0. The genes that the fragments are predicted to have been amplified from are also indicated. θ, an uncharacterized locus in E. coli bearing sequence similarity to characterized loci in other bacteria; ϕ, no matches were found, indicating a noncoding region; ∗, hypothetical, as yet uncharacterized genes.

FIG. 2

Distance tree of the EcoR collection of strains and 15 serogroup O157 strains obtained by the FAFLP assay. EcoR strains are labelled 01 to 72. The tree was generated by using a matrix of pairwise distances calculated for all strains with the coefficient of Nei and Li (Dice) (18). The MLEE groupings of the EcoR collection and the O157 VTEC group are circled. The VT-negative strains of serotypes O157:H8 (strain 74850), O157:H19 (strain 10964), and O157:H42 (strain 73886) that did not cluster with the O157 VTEC group are boxed in black. The nine EcoR strains that did not cluster with their original MLEE groupings are boxed in grey and are followed by their MLEE group designation.

FIG. 3

Genotyper FAFLP assay output for four O157 serotypes representing the diversity of O157: O157:H7 (strain 12900), O157:H19 (strain 10964), O157:H8 (strain 74850), and O157:H42 (strain 73886). The boxed numbers under the peaks of the traces are the fragment sizes assigned by Genotyper after comparison with the standard curve generated with the internal size standard.