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.
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 (alpha1, alpha2, beta1, beta2, gamma1, gamma2/theta, delta/kappa, epsilon, zeta, eta, iota, lambda, mu, nu, xi, omicron) of the eae gene, 4 variants (alpha1, beta1, gamma1, gamma2/theta) of the tir gene, 4 variants (alpha1, beta1, beta2, gamma1) of the espA gene, 3 variants (alpha1, beta1, gamma1) of the espB gene, and 3 variants (alpha1, beta1, gamma1) 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.
Garrido
Patricia
P
Laboratorio de Ecología Molecular, Centro de Astrobiología (INTA-CSIC), Madrid, Spain.
Blanco
Miguel
M
Moreno-Paz
Mercedes
M
Briones
Carlos
C
Dahbi
Ghizlane
G
Blanco
Jesús
J
Blanco
Jorge
J
Parro
Víctor
V
D000818:Animals; D020895:Enterocytes / Q000382:microbiology; D004926:Escherichia coli / Q000145:classification / Q000235:genetics / Q000472:pathogenicity; D029968:Escherichia coli Proteins / Q000235:genetics*; D005798:Genes, Bacterial; D014644:Genetic Variation; D006801:Humans; D020411:Oligonucleotide Array Sequence Analysis; D010750:Phosphoproteins / Q000235:genetics*; D012703:Serotyping / Q000379:methods; D022603:Shiga Toxins / Q000096:biosynthesis*; D014774:Virulence
D029968:Escherichia coli Proteins; C457340:LEE protein, E coli; D010750:Phosphoproteins; D022603:Shiga Toxins