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Clinical Chemistry 52: 192-201, 2006. First published December 29, 2005; 10.1373/clinchem.2005.059766
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(Clinical Chemistry. 2006;52:192-201.)
© 2006 American Association for Clinical Chemistry, Inc.

Molecular Diagnostics and Genetics

STEC-EPEC Oligonucleotide Microarray: A New Tool for Typing Genetic Variants of the LEE Pathogenicity Island of Human and Animal Shiga Toxin–Producing Escherichia coli (STEC) and Enteropathogenic E. coli (EPEC) Strains

Patricia Garrido1, Miguel Blanco2, Mercedes Moreno-Paz1, Carlos Briones1, Ghizlane Dahbi2, Jesús Blanco2, Jorge Blanco2 and Víctor Parro1,a

1 Laboratorio de Ecología Molecular, Centro de Astrobiología (INTA-CSIC), Madrid, Spain.
2 Laboratorio de Referencia de E. coli (LREC), Departamento de Microbiología y Parasitología, Facultad de Veterinaria, Universidad de Santiago de Compostela, Lugo, Spain.

aAddress correspondence to this author at: Laboratorio de Ecología Molecular, Centro de Astrobiología (INTA-CSIC), carretera de Ajalvir km 4, 28850, Torrejón de Ardoz, Madrid, Spain. Fax 34-915201074; e-mail parrogv{at}inta.es.

Background: Shiga toxin–producing Escherichia coli (STEC) and enteropathogenic E. coli (EPEC) are important emerging pathogens that can cause a severe and sometimes fatal illness. Differentiation of eae, tir, espA, espD, and espB gene variants of the locus of enterocyte effacement (LEE) pathogenicity island represents an important tool for typing in routine diagnostics as well as in pathogenesis, epidemiologic, clonal, and immunologic studies.

Methods: Type-specific oligonucleotide microarrays and a PCR scheme were designed and constructed for the detection and typing of genetic variants of the LEE genes. Oligonucleotide probes were tested for their specificity against the corresponding type strain by microarray hybridization using fluorescent DNA, either PCR-amplified (single, multiplex, long-range), chromosomal, or amplified chromosomal DNA.

Results: The PCR scheme and the oligonucleotide microarray allowed us to distinguish 16 variants ({alpha}1, {alpha}2, ß1, ß2, {gamma}1, {gamma}2/{theta}, {delta}/{kappa}, {epsilon}, {zeta}, {eta}, {iota}, {lambda}, µ, {nu}, {xi}, o) of the eae gene, 4 variants ({alpha}1, ß1, {gamma}1, {gamma}2/{theta}) of the tir gene, 4 variants ({alpha}1, ß1, ß2, {gamma}1) of the espA gene, 3 variants ({alpha}1, ß1, {gamma}1) of the espB gene, and 3 variants ({alpha}1, ß1, {gamma}1) of the espD gene. We found a total of 12 different combinations of tir, espA, espB, and espD genes among the 25 typed strains.

Conclusions: The PCR scheme and the oligonucleotide microarray described are effective tools to rapidly screen multiple virulence genes and their variants in E. coli strains isolated from human and animal infections. The results demonstrate the great genetic diversity among LEE genes of human and animal STEC and EPEC strains.




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