Detection of virulent Escherichia coli O157 strains using multiplex PCR and single base sequencing for SNP characterization

Abstract

Aims: To compare 167 Norwegian human and nonhuman Escherichia coli O157:H7/NM (nonmotile) isolates with respect to an A/T single nucleotide polymorphism (SNP) in the tir gene and to detect specific SNPs that differentiate STEC O157 into distinct virulence clades (1–3 and 8). Methods and Results: We developed a multiplex PCR followed by single base sequencing for detection of the SNPs, and examined the association among SNP genotype, virulence profile (stx and eae status), multilocus variable number of tandem repeats analysis (MLVA) profile and clinical outcome. We found an over‐representation of the T allele among human strains compared to nonhuman strains, including 5/6 haemolytic‐uraemic syndrome cases. Fourteen strains belonged to clade 8, followed by two clade 2 strains. No clade 1 nor 3 isolates were observed. stx1 in combination with either stx2EDL933 or stx2c were frequently observed among human strains, whereas stx2c was dominating in nonhuman strains. MLVA indicated that only single cases or small outbreaks with E. coli O157 have been observed in Norway through the years 1993–2008. Conclusion: We observed that the tir‐255 A/T SNP and the stx status were different between human and nonhuman O157 strains. No major outbreaks were observed, and only a few strains were differentiated into the virulence clades 2 and 8. Significance and Impact of the Study: The detection of virulence clade‐specific SNPs enables the rapid designation of virulent E. coli O157 strains, especially in outbreak situations.

Genetic Features Differentiating Bovine, Food, and Human Isolates of Shiga Toxin-Producing Escherichia coli O157 in The Netherlands

“…Consequently, like with the Q gene variants, bovine isolates were dominated by isolates carrying only stx 2c (86%), while this frequency was only 37% among clinical isolates. Similar patterns were reported by recent Norwegian (18) and Japanese (26) studies. Epidemiological studies have revealed that stx 2a is more strongly associated with clinical isolates and causes more severe symptoms than stx 2c (1,4,21,36).…”

“…The results of the tir(A255T) polymorphism assay confirm earlier findings of an overrepresentation of the tir(255T) variant among human clinical isolates of STEC O157 isolates and an approximately equal distribution of both variants among bovine isolates (6,18). The reason for the specific association of human clinical isolates with tir(255T) is unknown.…”

Molecular Typing of Escherichia coli O157:H7 Isolates from Swedish Cattle and Human Cases: Population Dynamics and Virulence

“…Argentina has a high clade 8 prevalence among E. coli O157:H7 in cattle (ϳ50%) and even higher among human cases (ϳ80%) (25), together with an extremely high HUS incidence (26) compared to countries with little or no clade 8 strains. A few studies on clade 8 have been performed in Europe; in a set of Norwegian isolates investigated by Haugum et al (27), clade 8 occurred more commonly in human than in nonhuman sources but was not significantly associated with HUS (27). A recent study comparing cattle and human isolates of E. coli O157:H7 from the Netherlands found no specific association of clade 8 with strains of human clinical origin (28).…”

Rapid Advances in Nucleic Acid Technologies for Detection and Diagnostics of Pathogens

“…The production of large numbers of low cost reads from these sequencing platforms makes these next generation sequencing technologies to be applied for quick de novo assemblies of out breaking pathogenic bacterial and viral genomes and a quick analysis to determine its identity as done for human influenza A viruses and virulent Escherichia coli O157 strains. 34,35 Moving forward these automated sequencing technologies will play a crucial role in detection and diagnostics of pathogens of future outbreaks.…”