Differentiation of Escherichia coli Pathotypes by Oligonucleotide Spotted Array

Abstract

To accurately determine the pathotypes of Escherichia coli strains, a comprehensive assessment of each strain that targets multiple genes is required. A new approach to the identification and characterization of E. coli pathotypes was developed by constructing gene-specific probes (70-mers) for not only the virulence genes associated with each E. coli pathotype but also the O157-, CFT073-, and K-12-specific and common genes of each pathotype. Analysis of oligonucleotide probes with reference and clinical isolates of E. coli pathotypes indicated that the array could differentiate the pathotypes on the basis of their virulence and specific gene patterns. Probes targeting common genes of E. coli were present in all the reference and clinical strains. Salmonella enterica subsp. enterica-specific genes and Salmonella core genes were used as negative controls. The entire E. coli pathotype showed reactivity to only 4 of the 81 Salmonella-specific gene probes. Characterization of the genetic and virulence profiles of a single strain by using probes for virulence factors and specific and common genes in the spotted array is an ideal diagnostic tool for determination of E. coli pathotypes and could also have a significant impact on the epidemiological analysis of E. coli infections.Escherichia coli is a normal commensal gram-negative rodshaped bacterium that lives inside the intestinal tracts of humans and warm-blooded animals. However, some E. coli strains cause urinary tract infections, bacteremia, and bacteriumrelated diarrhea and are also the main cause of neonatal meningitis in human and animals. In 1999, the Centers for Disease Control and Prevention estimated that there were 269,060 cases of gastroenteritis caused by E. coli in the United States alone (15). Pathogenic E. coli strains can be distinguished from their nonpathogenic counterparts by the presence of virulence genes, which code for adherence and colonization, invasion, cell surface molecules, secretion, transport, and siderophore formation (9). These virulence genes are generally organized as large blocks in chromosomes, plasmids, or phages and are often transmissible between E. coli strains. Based on the type of virulence factor present and host clinical symptoms, E. coli strains are categorized into pathotypes: (i) enteropathogenic E. coli (EPEC), which causes diarrhea in children and animals; (ii) enterohemorrhagic E. coli (EHEC), which is responsible for hemorrhagic colitis and hemolytic-uremic syndrome; (iii) enterotoxigenic E. coli (ETEC), which causes traveler's diarrhea and porcine and bovine diarrhea; (iv) enteroaggregative E. coli (EAEC), which causes persistent diarrhea in humans, and diffusely adherent E. coli (DAEC), a subclass of enteroaggregative E. coli which causes diarrhea in children; (v) enteroinvasive E. coli (EIEC), which causes watery diarrhea and dysentery; (vi) uropathogenic E. coli (UPEC), which causes urinary tract infections in humans and animals; and (vii) neonatal meningitis E. coli (NMEC), which is responsible for meningitis...

Identification of Escherichia coli Genes Associated with Urinary Tract Infections

“…Two micrograms of the genomic DNA was used for the enzyme digestion and labeling processes and then concentrated to a volume of 12 l for the following hybridization with the microarray. Microarray printing, bacterial genomic DNA labeling, microarray hybridization, and data acquisition were performed as previously described (27). Each slide had triplicate spots of each gene.…”

“…The remaining genes are derived from other E. coli strains capable of causing intestinal infections. The association of the majority of the 342 genes in the array with UTIs has not yet been investigated, except for 36 uropathogenic genes included among them (27).…”

“…Palaniappan et al have developed an oligonucleotide-spotted microarray containing probes representing 342 E. coli genes to differentiate E. coli pathotypes (27). A majority of the genes are derived from the UPEC strain CFT073, the enterohemorrhagic E. coli strain EDL933, and the commensal E. coli K-12 strain MG1655.…”

Potentials and limitations of molecular diagnostic methods in food safety

“…Another approach exploits over 400 long DNA oligos (70mers) to detect and classify 11 different E. coli pathotypes, i.e. groups of strains hosting similar virulence factor and causing similar clinical symptoms [44]. The assay relies on pathotype-specific genes as well as E. coli core sequences.…”

Genotypic analysis of Escherichia coli strains that cause urosepsis in the Aegean region

“…coli strains that cause diseases in systems other than the intestinal tract, such as the urinary or circulatory systems, are called extraintestinal pathogenic E. coli (ExPEC), and these strains can be divided into four groups: uropathogenic E. coli (UPEC), neonatal-meningitis-causing E. coli, avian pathogenic E. coli (APEC), and sepsis pathogenic E. coli (1)(2)(3)(4). Although ExPEC strains are the most common cause of urinary tract infections, they might be the pathogens of sepsis, neonatal meningitis, and pneumonia.…”