Fast DNA serotyping of Escherichia coli by use of an oligonucleotide microarray

Abstract

Classical antibody-based serotyping of Escherichia coli is an important method in diagnostic microbiology for epidemiological purposes, as well as for a rough virulence assessment. However, serotyping is so tedious that its use is restricted to a few reference laboratories. To improve this situation we developed and validated a genetic approach for serotyping based on the microarray technology. The genes encoding the O-antigen flippase (wzx) and the O-antigen polymerase (wzy) were selected as target sequences for the O antigen, whereas fliC and related genes, which code for the flagellar monomer, were chosen as representatives for the H phenotype. Starting with a detailed bioinformatic analysis and oligonucleotide design, an ArrayTube-based assay was established: a fast and robust DNA extraction method was coupled with a site-specific, linear multiplex labeling procedure and hybridization analysis of the biotinylated amplicons. The microarray contained oligonucleotide DNA probes, each in duplicate, representing 24 of the epidemiologically most relevant of the over 180 known O antigens (O antigens 4, 6 to 9, 15, 26, 52, 53, 55, 79, 86, 91, 101, 103, 104, 111, 113, 114, 121, 128, 145, 157, and 172) as well as 47 of the 53 different H antigens (H antigens 1 to 12, 14 to 16, 18 to 21, 23 to 34, 37 to 43, 45, 46, 48, 49, 51 to 54, and 56). Evaluation of the microarray with a set of defined strains representing all O and H serotypes covered revealed that it has a high sensitivity and a high specificity. All of the conventionally typed 24 O groups and all of the 47 H serotypes were correctly identified. Moreover, strains which were nonmotile or nontypeable by previous serotyping assays yielded unequivocal results with the novel ArrayTube assay, which proved to be a valuable alternative to classical serotyping, allowing processing of single colonies within a single working day.

FIG. 1.

Layout of the oligonucleotide probes on the microarray. Details are given in Table 2.

FIG. 2.

Microphotographs of microarrays hybridized with genomic DNA of E. coli O157:H7 (A), O53:H3 (B), and O172:K−:H− (C). The numbers correspond to the spot identifiers given in Table 2 and Fig. 1.

FIG. 3.

Microphotographs of microarrays of E. coli O157:H43 for which 1 μg of genomic DNA (A), 3 μl of a single colony lysate with a 100-μl total volume (B), and 3 μl of a single colony resuspended in 100 μl water (C) was used for labeling. The numbers correspond to the spot identifiers given in Table 2 and Fig. 1.