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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.

SUBMITTER: Bekal S

PROVIDER: S-EPMC154688 | biostudies-literature | 2003 May

REPOSITORIES: biostudies-literature

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

Bekal Sadjia S Brousseau Roland R Masson Luke L Prefontaine Gabrielle G Fairbrother John J Harel Josée J

Journal of clinical microbiology 20030501 5

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 ...[more]

PMID: 12734257

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Project description:BackgroundEscherichia coli is a normal inhabitant of the gut which upon acquiring virulence factors becomes potentially able to cause diseases. Although E. coli population augments in Crohn's disease (CD), the reason of this proliferation is not yet clear. CD associated E. coli shows features of extraintestinal pathogenic categories (ExPEC), and eventually the ability to invade cultured epithelial cells, a property observed among diarrheagenic E. coli (DEC). In this work, data on the characterization of an E. coli isolate from a CD patient reveal that, besides invasiveness, CD associated E. coli may harbor other typical DEC markers, namely those defining enterohemorragic (EHEC) and enteroaggregative (EAEC) pathotypes.ResultsThe studied strain, detected both in an ileum biopsy and stools, belonged to the B2 E. coli reference collection (EcoR) phylogroup and harbored the intimin, Shiga cytotoxin 1, and AggR transcriptional activator encoding genes (eae, stx1, aggR, respectively); displayed aggregative adherence to Hep-2 cells and an ability to enter Caco-2 cells four times as high as that of EIEC reference strain and half of invasiveness of AIEC LF82. It was able to enter and replicate in J774 macrophages with invasiveness 85 times as high as that of LF82, but with only one sixth of the intracellular proliferation ability of the later. Extracellular products with cytotoxic activity on Vero cells were detected in strain's cultures. Preliminary analysis indicated similarity of this strain's genome with that of O104:H4/2011C-3493.MethodsFollowing its isolation from a resected CD patient, the strain was characterized by in vitro adhesion and invasion assays to Hep-2, invasion to Caco-2 cells and to J774 macrophages and tested for the ability to form biofilm and to produce Shiga cytotoxins. PCRs were carried out to identify virulence genetic markers and for EcoR phylogrouping. The strain's genome was sequenced by means of Ion torrent PGM platform.ConclusionThe detection, in a CD patient, of an E. coli combining virulence features of multiple DEC pathotypes seems not only to stress the relevance of E. coli to CD etiopathogenesis but also to indicate the existence of new and potentially more virulent strains putatively associated with this disease.

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

Publications

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

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