Rapid identification of Escherichia coli pathotypes by virulence gene detection with DNA microarrays

Abstract

One approach to the accurate determination of the pathogenic potential (pathotype) of isolated Escherichia coli strains would be through a complete assessment of each strain for the presence of all known E. coli virulence factors. To accomplish this, an E. coli virulence factor DNA microarray composed of 105 DNA PCR amplicons printed on glass slides and arranged in eight subarrays corresponding to different E. coli pathotypes was developed. Fluorescently labeled genomic DNAs from E. coli strains representing known pathotypes were initially hybridized to the virulence gene microarrays for both chip optimization and validation. Hybridization pattern analysis with clinical isolates permitted a rapid assessment of their virulence attributes and determination of the pathogenic group to which they belonged. Virulence factors belonging to two different pathotypes were detected in one human E. coli isolate (strain H87-5406). The microarray was also tested for its ability to distinguish among phylogenetic groups of genes by using gene probes derived from the attaching-and-effacing locus (espA, espB, tir). After hybridization with these probes, we were able to distinguish E. coli strains harboring espA, espB, and tir sequences closely related to the gene sequences of an enterohemorrhagic strain (EDL933), a human enteropathogenic strain (E2348/69), or an animal enteropathogenic strain (RDEC-1). Our results show that the virulence factor microarray is a powerful tool for diagnosis-based studies and that the concept is useful for both gene quantitation and subtyping. Additionally, the multitude of virulence genes present on the microarray should greatly facilitate the detection of virulence genes acquired by horizontal transfer and the identification of emerging pathotypes.

FIG. 1.

Print pattern of the E. coli pathotype microarray indicated by grouping of genes by category and locations of the individual genes.

FIG. 2.

Detection of virulence genes and simultaneous identification of the pathotypes of known E. coli strains after microarray hybridization with genomic DNA from nonpathogenic K-12 strain E. coli DH5α (A), enterohemorrhagic strain EDL933 O157:H7 (B), uropathogenic strain J96 O4:K6 (C), and enterotoxigenic strain H-10407 (D). After HindIII-EcoRI digestion, genomic DNA was labeled with Cy3. Labeled DNA (500 ng) was hybridized to the array overnight at 42°C, washed, dried, and scanned. The boxed spots in panel A represent the virulence genes present in E. coli K-12 strain DH5α (traT, fimA, fimH, ompA, ompT, iss, and fliC). The boxed spots in panels B, C, and D indicate the pathotype-specific genes in the strains tested. Genes present in more than one pathotype (iss, irp2, fliC, and ompT) or present in all pathotypes (fimH, fimA, and ompA) gave positive signals. The horizontal bar indicates the color representation of the fluorescence signal intensity.

FIG. 3.

Analysis of virulence potential of E. coli strains isolated from clinical samples by using the E. coli pathotype microarray. (A) Hybridization pattern obtained with genomic DNA from avian E. coli isolate Av01-4156; (B) hybridization pattern obtained with genomic DNA from bovine strain B00-4830; (C) hybridization obtained with genomic DNA from human E. coli isolate H87-540. Labeled DNA (500 ng) was hybridized to the array overnight at 42°C, after which the slide was washed, dried, and scanned. Boxed spots indicate pathotype-specific genes iucD, iron, traT, and iutA in panel A; pathotype-specific genes etpD, F5, stap, and traT in panel B; and pathotype-specific genes stx₁, cdt2, cdt3, afaD8, bmaE, iucD, iroN, and iutA in panel C. Positive signals were also obtained with genes present in more than one pathotype (espP, iss, ompT, and fliC) and genes present in all the pathotypes tested (fimA, fimH, and ompA).

FIG. 4.

Detection of stx and cnf variant genes in clinical isolates of E. coli by use of the pathotype microarray. The white boxes in panel A outline the stx genes hybridized with human strain H87-5406 (row 1) and bovine strain B99-4297 (row 2). The white boxes in panel B outline the cnf genes hybridized with strain Ca01-E179 (row 1) and strain H87-5406 (row 2). Labeled DNA (500 ng) was hybridized to an array overnight at 42°C, after which the slide was washed, dried, and scanned.

FIG. 5.

Use of the E. coli pathotype microarray to identify the phylogenetic groups of E. coli strains on the basis of their hybridization patterns with the attaching-and-effacing gene probes. (A) Print patterns obtained with the espA, espB, and tir probes on the pathotype microarray, with the homology percentages between each immobilized probe indicated; (B) detection of espA3, espB2, and tir3 in human EPEC strain E2348/69; (C) hybridization pattern obtained with genomic DNA from animal EPEC strain P86-1390 (espA1, espB3, and tir1); (D) detection of espA2, espB1, and tir2 in EHEC strain EDL933. The positive hybridization results obtained with the espA, espB, and tir probes are outlined in white boxes.