Use of the espZ gene encoded in the locus of enterocyte effacement for molecular typing of shiga toxin-producing Escherichia coli

Abstract

Infections with Shiga toxin-producing Escherichia coli (STEC) result in frequent cases of sporadic and outbreak-associated enteric bacterial disease in humans. Classification of STEC is by stx genotype (encoding the Shiga toxins), O and H antigen serotype, and seropathotype (subgroupings based upon the clinical relevance and virulence-related genotypes of individual serotypes). The espZ gene is encoded in the locus of enterocyte effacement (LEE) pathogenicity island responsible for the attaching and effacing (A/E) lesions caused by various E. coli pathogens (but not limited to STEC), and this individual gene ( approximately 300 bp) has previously been identified as hypervariable among these A/E pathogens. Sequence analysis of the espZ locus encoded by additional STEC serotypes and strains (including O26:H11, O121:H19, O111:NM, O145:NM, O165:H25, O121:NM, O157:NM, O157:H7, and O5:NM) indicated that distinct sequence variants exist which correlate to subgroups among these serotypes. Allelic discrimination at the espZ locus was achieved using Light Upon eXtension real-time PCR and by liquid microsphere suspension arrays. The allele subtype of espZ did not correlate with STEC seropathotype classification; however, a correlation with the allele type of the LEE-encoded intimin (eae) gene was supported, and these sequence variations were conserved among individual serotypes. The study focused on the characterization of three clinically significant seropathotypes of LEE-positive STEC, and we have used the observed genetic variation at a pathogen-specific locus for detection and subtyping of STEC.

FIG. 1.

Relatedness of espZ carried by STEC. (A) Multiple sequence alignment of the espZ central variable region, with LUX primers specific for individual espZ subtypes indicated by arrows (shown directly above the corresponding sequences; the arrowheads represent the 3′ end of the primers) and liquid microsphere suspension probes indicated by lines with spheres (the latter representing the 5′ end of the oligonucleotides where the microsphere is covalently attached). See Table 2 for the nucleotide sequence of these primers and probes. The double-bracketed line indicates the intervening loop-encoding region between the two surrounding predicted transmembrane domain-encoding regions (not indicated). Although sequences are broken into two sections asymmetrically, each of the espZ sequences is contiguous from the top to the bottom panel. The corresponding espZ allele subtype, serotype, and strain number for each sequence are indicated. (B) Phylogenetic distance between espZ encoded by different STEC serotypes represented by a neighbor-joining tree, and the scale bar indicates distance scores. Strain identification numbers are indicated in brackets. The espZ allele subtypes, corresponding to each cluster, are indicated by curly brackets. Nucleotide accession numbers are included in Materials and Methods and the text.

FIG. 2.

Real-time LUX PCR using allele-specific espZ primers. (A) The espZ-γ2 LUX primers were used on a panel of strains including three O111:NM isolates and a representative strain from other LEE-positive and -negative STEC (serotypes and respective strain numbers are indicated in the insets). (B) Detection limit testing on a dilution series of purified O111:NM genomic DNA. DNA amounts indicated in the legend represent the total amount of purified DNA present in the reaction tube. The horizontal line indicates the threshold value of 30 fluorescent units used to determine positive reactions. NTC, no template control.

FIG. 3.

Microsphere suspension array allelic discrimination of espZ encoded by different STEC serotypes. Biotin-labeled espZ template was amplified from strains of the indicated serotypes and incubated with a mixture of four fluorescently coded microspheres coupled with an oligonucleotide probe targeting the four espZ allele subtypes (see inset). The background fluorescence contributed by the microsphere-probe mixture was determined by using a no-template control (TE buffer), and standard errors are indicated by a vertical line on each bar.

FIG. 4.

Split decomposition analysis of espZ. Recombination between loci is indicated when the topology resembles a network rather than the single branching points seen in normal tree topologies. Strain identifications are indicated in brackets. Nucleotide accession numbers are included in Materials and Methods and the text.